Makrolife Biotech Seal: Testing Procedures and Certification Overview

At Makrolife Biotech GmbH, we provide a unique seal of approval for compounds that demonstrate outstanding safety, efficacy, and immunological compatibility. Our rigorous testing procedures are designed to evaluate the effects of compounds on the human immune system, particularly macrophages, using state-of-the-art technologies and scientific methodologies. Below, we outline the steps and criteria used to award the Makrolife Biotech Seal. Our laboratories are located in Bruchsal at Triwo Park Technologypark.

Introduction to Makrolife Biotech

Makrolife Biotech GmbH is a leading innovator in the field of immunological testing, specializing in the evaluation of how compounds interact with the human immune system. By combining advanced scientific methodologies with cutting-edge technology, we aim to ensure the safety and efficacy of products while promoting public health. Our commitment to excellence has established us as a trusted partner for companies seeking reliable immunological evaluations.

 

The testing mathods of Makrolife Biotech to get a Makrolife Seal.

These are internal testing methods

Below is a detailed Materials and Methods section for isolating monocytes and macrophages from human blood, including steps for obtaining them from whole blood or commercially.

Materials and Methods

1. Materials

• Human Blood Source
• Fresh whole blood collected in heparin or EDTA tubes (source: healthy donors or blood banks).
• Alternatively, Peripheral Blood Mononuclear Cells (PBMCs) can be purchased (e.g., from companies like Lonza, Stemcell Technologies, BioIVT).
• Reagents
• Ficoll-Paque™ Plus (density gradient medium, GE Healthcare or Cytiva).
• Phosphate-Buffered Saline (PBS) without calcium and magnesium.
• EDTA (0.5M stock solution).
• Cell culture media (e.g., RPMI 1640 or DMEM, supplemented with 10% fetal bovine serum [FBS] and 1% Penicillin-Streptomycin).
• Recombinant human macrophage colony-stimulating factor (M-CSF, e.g., from PeproTech or R&D Systems) for macrophage differentiation.
• Plasticware
• 50 mL conical tubes.
• Sterile pipette tips.
• 6-well culture plates (or other culture vessels).
• Magnetic Isolation
• Monocyte isolation kits (e.g., EasySep™ Human Monocyte Isolation Kit from Stemcell Technologies or CD14 MicroBeads from Miltenyi Biotec).
• Magnetic separator for microbeads.

2. Methods

2.1 Isolation of PBMCs from Whole Blood

1. Blood Collection
   Collect fresh blood in sterile tubes containing anticoagulant (e.g., heparin or EDTA). Blood should be processed within 4–6 hours of collection to maintain cell viability.
2. Density Gradient Centrifugation

• Dilute blood 1:1 with sterile PBS (e.g., 20 mL blood + 20 mL PBS).
• Slowly layer diluted blood onto an equal volume of Ficoll-Paque™ in a 50 mL conical tube without mixing.
• Centrifuge at 400 x g for 30–40 minutes at room temperature (no brake).

1. PBMC Layer Collection

• After centrifugation, three layers will form:
• Top: plasma.
• Middle: PBMC layer (buffy coat).
• Bottom: Ficoll-Paque and erythrocytes.
• Carefully aspirate the PBMC layer using a sterile pipette and transfer to a new 50 mL tube.

1. Washing PBMCs

• Add PBS (2–3x the volume of PBMCs), centrifuge at 300 x g for 10 minutes, and discard the supernatant. Repeat the wash 2–3 times.
• Resuspend the pellet in complete cell culture media.

2.2 Isolation of Monocytes from PBMCs

1. Magnetic Isolation

• Resuspend PBMCs in buffer provided by the monocyte isolation kit (e.g., MACS buffer or PBS with 0.5% BSA and 2 mM EDTA).
• Add the CD14 MicroBeads or monocyte isolation cocktail as per the manufacturer’s protocol.
• Incubate at 4°C for 15–30 minutes with gentle mixing.
• Wash the cells with buffer and centrifuge at 300 x g for 5 minutes.
• Pass the cell suspension through a magnetic separator to isolate CD14+ monocytes.

1. Confirmation of Monocyte Purity

• Confirm purity via flow cytometry (e.g., CD14 marker) or microscopy.

2.3 Differentiation of Monocytes into Macrophages

1. Culture Setup

• Plate monocytes in 6-well plates at 1 × 10⁶ cells/mL in RPMI 1640 or DMEM supplemented with 10% FBS.
• Add M-CSF (50 ng/mL) to the culture medium to promote macrophage differentiation.

1. Incubation

• Incubate at 37°C in 5% CO₂ for 5–7 days. Refresh the medium every 2–3 days with fresh M-CSF.

1. Morphological Verification

• Observe cells under a microscope. Macrophages will appear adherent, flattened, and larger compared to monocytes.

3. Sources to Purchase PBMCs or Monocytes

If fresh blood collection is not feasible, PBMCs or monocytes can be purchased from:

1. Lonza: Offers cryopreserved PBMCs or CD14+ monocytes.
2. Stemcell Technologies: Provides frozen PBMCs and EasySep™ kits for monocyte isolation.
3. BioIVT: Supplies isolated PBMCs and monocytes from healthy and diseased donors.
4. AllCells: Offers primary cells from a range of donors.
5. Miltenyi Biotec: Sells monocyte isolation reagents and magnetic cell sorting kits.

 

 

Isolated and PMCS are finalized and can be further used to co-incubate with specific compound

We are using 1000 macrophages for each testing in a triplicate to gain reproductive results.

 

Below is a comprehensive Materials and Methods section for conducting cytokine tests (ELISA or multiplex tests) to measure immune reactions triggered in monocytes and macrophages by co-incubating a specific compound.

Materials and Methods

1. Materials

1.1 Cells and Media

• Monocytes and Macrophages:
• Isolated from fresh human blood as described in the previous method or purchased (e.g., from Lonza, Stemcell Technologies, or BioIVT).
• If macrophages are needed, differentiate monocytes by culturing with M-CSF (50 ng/mL) for 5–7 days.
• Culture Media:
• RPMI 1640 or DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% Penicillin-Streptomycin, and 2 mM L-glutamine.

1.2 Reagents

• Test Compound:
• Pharmaceutical or other compound under study, dissolved in an appropriate solvent (e.g., DMSO or PBS).
• Lipopolysaccharide (LPS):
• Positive control to trigger an immune reaction (e.g., from Escherichia coli O111:B4, Sigma-Aldrich).
• Negative Control:
• Vehicle control (e.g., 0.1% DMSO or PBS matching the solvent for the test compound).
• Cytokine Assay Kits:
• ELISA Kits:
• For IL-6, TNF-α, IL-1β, IL-10, and other cytokines (e.g., Thermo Fisher Scientific, BD Biosciences, or R&D Systems).
• Multiplex Cytokine Kits:
• Bead-based immunoassays for simultaneous cytokine quantification (e.g., Bio-Plex™ from Bio-Rad or Luminex™ from Thermo Fisher).
• PBS:
• Without calcium and magnesium for washes.

1.3 Plasticware and Consumables

• Sterile 96-well flat-bottom plates (for cell culture).
• ELISA plates (if using single-plex assays).
• Multiplex-compatible plates (if using Luminex/Bio-Plex).
• Pipettes and sterile filter tips.
• Cell scraper (optional, if required to detach cells).

1.4 Equipment

• CO₂ incubator (37°C, 5% CO₂).
• Microplate reader for ELISA.
• Multiplex assay analyzer (e.g., Luminex™ 200 system or similar).
• Centrifuge with a rotor for 96-well plates.
• Inverted microscope (to monitor cell morphology).

2. Methods

2.1 Cell Preparation

1. Monocyte Isolation:

• Isolate monocytes as described earlier or purchase cryopreserved PBMCs/monocytes and thaw according to manufacturer instructions.

1. Macrophage Differentiation:

• Plate monocytes in RPMI 1640 supplemented with 10% FBS and M-CSF (50 ng/mL).
• Incubate at 37°C and 5% CO₂ for 5–7 days, replacing media every 2–3 days.

2.2 Experimental Setup

1. Plating Cells:

• Seed monocytes/macrophages in 96-well plates at a density of 1 × 10⁵ cells/well in 200 µL of complete culture medium.
• Allow macrophages to adhere overnight before treatment.

1. Compound Treatment:

• Prepare test compound dilutions in culture medium.
• Add test compound to wells at the desired concentrations (e.g., 1 nM to 100 µM, depending on experimental design).
• Include the following controls:
• Positive Control: Add LPS (final concentration: 1 ng/mL to 100 ng/mL).
• Negative Control: Add the vehicle (e.g., 0.1% DMSO or PBS).

1. Co-Incubation:

• Incubate cells with the test compound for 24 hours at 37°C and 5% CO₂.

2.3 Cytokine Collection and Analysis

1. Supernatant Collection:

• After 24 hours, carefully collect the culture supernatants (without disturbing cells) and transfer to sterile tubes or plates.
• Centrifuge at 300 x g for 5 minutes to remove debris.
• Store supernatants at -80°C if not analyzing immediately.

1. Cytokine Quantification:

• ELISA:
• Perform ELISA for individual cytokines (e.g., IL-6, TNF-α, IL-1β, and IL-10) according to the manufacturer’s protocol.
• Read absorbance at the recommended wavelength (e.g., 450 nm) using a microplate reader.
• Multiplex Cytokine Assay:
• Use a multiplex bead-based assay to measure multiple cytokines simultaneously.
• Prepare samples and standards according to the manufacturer’s protocol.
• Analyze samples using a Luminex analyzer.

2.4 Normalization and Data Analysis

1. Normalization:

• Normalize cytokine levels to cell number (e.g., cytokine concentration per 1 × 10⁵ cells).
• For multiplex assays, ensure appropriate gating and standard curve fitting.

1. Statistical Analysis:

• Perform statistical tests (e.g., ANOVA or t-test) to compare cytokine levels between test compound, positive control, and negative control groups.

3. Notes and Considerations

• Ensure the solvent used for the test compound does not exceed 0.1% in the culture medium, as higher concentrations can be cytotoxic.
• Include triplicates or quadruplicates for each condition to ensure reproducibility.
• Monitor cell morphology to ensure test compound or controls do not induce cell death.
• Use low-endotoxin reagents to prevent unintended activation of immune responses.

4. Vendors for Reagents and Kits

• Cytokine ELISA Kits: Thermo Fisher Scientific, BD Biosciences, R&D Systems.
• Multiplex Assays: Bio-Rad (Bio-Plex), Thermo Fisher (ProcartaPlex), or MilliporeSigma (Milliplex).
• Recombinant M-CSF: PeproTech, BioLegend, or R&D Systems.
• Monocytes: Lonza, Stemcell Technologies, BioIVT, or AllCells.

 

 

 

 

Here is a Materials and Methods section for conducting a phagocytosis assay with monocytes and macrophages. The protocol includes co-incubation with a specific compound to measure immune reaction and phagocytic activity.

Materials and Methods

1. Materials

1.1 Cells and Culture Media

• Monocytes and Macrophages:
• Isolated from human blood as described previously or purchased (e.g., from Lonza, BioIVT, or Stemcell Technologies).
• Differentiated macrophages using M-CSF (50 ng/mL) for 5–7 days if required.
• Culture Media:
• RPMI 1640 or DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% Penicillin-Streptomycin, and 2 mM L-glutamine.

1.2 Phagocytosis Assay Reagents

• Fluorescent Particles:
• Fluorescently labeled zymosan particles, E. coli, or latex beads (e.g., Thermo Fisher Scientific or Sigma-Aldrich).
• Test Compound:
• Pharmaceutical or other compound dissolved in an appropriate solvent (e.g., DMSO or PBS).
• Controls:
• Positive Control: Lipopolysaccharide (LPS, 100 ng/mL, Sigma-Aldrich) or phorbol myristate acetate (PMA, 10 ng/mL, Sigma-Aldrich).
• Negative Control: Vehicle control (e.g., 0.1% DMSO or PBS).

1.3 Detection Reagents

• Trypan Blue Solution (0.4%):
• For quenching extracellular fluorescence during phagocytosis assays.
• Fixation Buffer:
• 4% paraformaldehyde in PBS.

1.4 Plasticware and Consumables

• Sterile 96-well flat-bottom plates.
• Pipettes and sterile tips.
• 15 mL and 50 mL centrifuge tubes.

1.5 Equipment

• CO₂ incubator (37°C, 5% CO₂).
• Fluorescence microscope or flow cytometer.
• Plate reader compatible with fluorescence detection.

2. Methods

2.1 Cell Preparation

1. Monocyte Isolation and Macrophage Differentiation:

• Isolate monocytes as described earlier.
• For macrophages, plate monocytes at 1 × 10⁶ cells/mL in RPMI 1640 with 10% FBS and M-CSF (50 ng/mL). Incubate at 37°C for 5–7 days, refreshing media every 2–3 days.

1. Plating for Assay:

• Seed monocytes or macrophages into 96-well plates at 1 × 10⁵ cells/well in 200 µL of complete culture media.
• Allow macrophages to adhere overnight.

2.2 Co-Incubation with Test Compounds

1. Preparation of Test Compound:

• Prepare dilutions of the test compound in culture media at desired concentrations (e.g., 1 nM to 100 µM).
• Include controls:
• Positive Control: LPS or PMA.
• Negative Control: Vehicle.

1. Treatment:

• Replace culture media in each well with 200 µL of media containing the test compound or controls.
• Incubate for 24 hours at 37°C and 5% CO₂.

2.3 Phagocytosis Assay

1. Preparation of Fluorescent Particles:

• Resuspend fluorescent zymosan particles, E. coli, or latex beads according to the manufacturer’s instructions.
• For zymosan or bacterial particles, opsonize them by incubating in 10% heat-inactivated human serum at 37°C for 30 minutes.

1. Adding Particles:

• Add fluorescent particles to the cells in a 10:1 particle-to-cell ratio.
• Incubate for 1 hour at 37°C and 5% CO₂ to allow phagocytosis.

1. Quenching Extracellular Fluorescence:

• After incubation, carefully wash wells three times with PBS to remove unengulfed particles.
• Add 0.2% trypan blue to each well for 2 minutes to quench extracellular fluorescence. Wash once with PBS.

1. Fixation:

• Add 4% paraformaldehyde to wells and incubate for 15 minutes at room temperature to fix cells.
• Wash wells with PBS to remove residual fixative.

2.4 Quantification

1. Fluorescence Microscopy:

• Visualize and quantify phagocytosed particles under a fluorescence microscope.
• Count the number of fluorescent particles per cell to measure phagocytic activity.

1. Flow Cytometry:

• Detach adherent cells (if using macrophages) with gentle scraping or cold PBS.
• Analyze fluorescence intensity using a flow cytometer to quantify the percentage of cells that have phagocytosed particles.

1. Plate Reader:

• If using a plate-based assay, measure fluorescence intensity with a plate reader (excitation and emission wavelengths appropriate for the particles).

2.5 Normalization and Data Analysis

1. Normalization:

• Normalize phagocytic activity to cell number (e.g., fluorescence intensity per 1 × 10⁵ cells).
• Subtract background fluorescence from wells without cells.

1. Statistical Analysis:

• Use statistical tests (e.g., ANOVA or t-tests) to compare phagocytosis rates across treatments and controls.

3. Notes and Considerations

1. Cytotoxicity Check:

• Test the compound for cytotoxicity using a viability assay (e.g., MTT or Trypan Blue exclusion) to ensure effects on phagocytosis are not due to cell death.

1. Serum-Free Incubation:

• Avoid serum in co-incubation media if the compound interacts with serum proteins.

1. Timepoints:

• For dynamic studies, measure phagocytosis at multiple time points (e.g., 30, 60, 120 minutes).

4. Vendors for Reagents and Kits

• Fluorescent Particles: Thermo Fisher Scientific, Sigma-Aldrich, Polysciences.
• Cytokine Stimulants: Sigma-Aldrich, Invivogen.
• Flow Cytometers and Plate Readers: BD Biosciences, Bio-Rad, Thermo Fisher Scientific.

 

Below is a comprehensive Materials and Methods section for conducting flow cytometry with monocytes and macrophages. The protocol includes co-incubation with a compound to trigger an immune reaction and measuring the immune response via flow cytometry.

Materials and Methods

1. Materials

1.1 Cells and Culture Media

• Monocytes and Macrophages:
• Isolated monocytes (from fresh blood or PBMCs, as described earlier) or purchased (e.g., from Lonza, Stemcell Technologies, or BioIVT).
• If macrophages are required, differentiate monocytes with M-CSF (50 ng/mL) for 5–7 days.
• Culture Media:
• RPMI 1640 or DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% Penicillin-Streptomycin, and 2 mM L-glutamine.

1.2 Stimulants and Test Compound

• Test Compound:
• Pharmaceutical or other compound, dissolved in DMSO or PBS.
• Controls:
• Positive Control: Lipopolysaccharide (LPS, 100 ng/mL) or phorbol myristate acetate (PMA, 10 ng/mL).
• Negative Control: Vehicle control (e.g., 0.1% DMSO or PBS).
• Fluorescent Antibodies for Flow Cytometry:
• Surface markers:
• CD14 (monocytes).
• CD68 (macrophages).
• HLA-DR (MHC-II) for activation status.
• CD80 and CD86 for co-stimulation.
• Intracellular markers (optional):
• Cytokines: IL-6, TNF-α, IL-10.
• Transcription factors (e.g., NF-κB for immune activation).
• Isotype Controls:
• Matched isotype antibodies for negative control.

1.3 Reagents

• Fixation/Permeabilization Buffer (e.g., BD Cytofix/Cytoperm, Thermo Fisher Scientific).
• FACS Buffer:
• PBS with 2% FBS or 0.5% BSA and 2 mM EDTA.
• Trypan Blue (optional for viability staining).
• Live/Dead Stain:
• Viability dye (e.g., Zombie Aqua™ from BioLegend or Live/Dead™ Fixable Stain from Invitrogen).
• Fc Block (optional):
• Human Fc Receptor Blocking Solution to minimize nonspecific binding.

1.4 Equipment

• Flow cytometer (e.g., BD FACSCanto II, LSRFortessa, or equivalent).
• CO₂ incubator (37°C, 5% CO₂).
• Centrifuge.

2. Methods

2.1 Cell Preparation

1. Monocyte Isolation and Macrophage Differentiation:

• Isolate monocytes or purchase cryopreserved cells.
• If macrophages are required, differentiate monocytes by plating in RPMI 1640 with 10% FBS and M-CSF (50 ng/mL) for 5–7 days.

1. Plating:

• Seed monocytes/macrophages in a 12-well plate at a density of 1 × 10⁶ cells/well in 2 mL of culture medium.
• Allow cells to adhere for at least 24 hours before treatment.

2.2 Co-Incubation with Test Compound

1. Preparation of Test Compound:

• Prepare serial dilutions of the test compound in culture media (e.g., 1 nM to 100 µM).
• Include controls:
• Positive Control: LPS or PMA.
• Negative Control: Vehicle.

1. Treatment:

• Replace media in each well with media containing the test compound, positive control, or negative control.
• Incubate cells at 37°C for 24 hours.

2.3 Staining for Flow Cytometry

1. Harvesting Cells:

• Gently detach adherent macrophages using cold PBS and cell scrapers or by incubation with 2 mM EDTA for 5 minutes at 37°C.
• For monocytes, transfer cells directly to 5 mL FACS tubes.
• Centrifuge at 300 x g for 5 minutes and discard the supernatant.

1. Viability Staining:

• Resuspend cells in 100 µL FACS buffer.
• Add 1 µL of viability dye (e.g., Zombie Aqua™) and incubate for 15 minutes in the dark at room temperature.
• Wash cells with 1 mL FACS buffer and centrifuge at 300 x g for 5 minutes.

1. Surface Staining:

• Resuspend cells in 100 µL FACS buffer.
• Add fluorescently labeled antibodies for surface markers (e.g., CD14, CD68, HLA-DR, CD80, CD86) at the recommended dilution.
• Include isotype controls.
• Incubate for 30 minutes at 4°C in the dark.

1. Intracellular Staining (Optional):

• Fix cells by adding 100 µL Fixation Buffer (e.g., BD Cytofix) and incubating for 15 minutes at room temperature.
• Permeabilize cells by washing with 1 mL Permeabilization Buffer and resuspend in 100 µL.
• Add intracellular antibodies (e.g., IL-6, TNF-α, IL-10) and incubate for 30 minutes at 4°C in the dark.

1. Final Wash:

• Wash cells twice with 1 mL FACS buffer.
• Resuspend in 300 µL FACS buffer for flow cytometry analysis.

2.4 Flow Cytometry Acquisition

1. Flow Cytometer Setup:

• Use appropriate laser and filter settings for the fluorophores.
• Run compensation controls and set gates using unstained cells, isotype controls, and single-stained controls.

1. Acquisition:

• Collect data for at least 10,000 events per sample.
• Use forward scatter (FSC) and side scatter (SSC) to identify monocytes/macrophages and exclude debris.

2.5 Data Analysis

1. Population Gating:

• Gate on viable, single cells (FSC-A vs. FSC-H).
• Identify monocytes (CD14+) or macrophages (CD68+).
• Assess activation markers (HLA-DR, CD80, CD86).

1. Cytokine Expression:

• For intracellular staining, quantify cytokines (e.g., IL-6, TNF-α) in gated populations.
• Compare cytokine production across conditions.

1. Statistical Analysis:

• Normalize data to the number of viable cells or fluorescence intensity per cell.
• Perform ANOVA or t-tests to determine significant differences between treatment groups.

3. Notes and Considerations

1. Titration of Antibodies:

• Optimize antibody concentrations for minimal background staining and maximal specificity.

1. Negative Controls:

• Include unstained, isotype, and single-stained controls for gating and compensation.

1. Timepoints:

• For dynamic studies, perform staining at multiple time points (e.g., 4, 8, 24 hours) to assess kinetics of immune activation.

1. Cytotoxicity Assay:

• Conduct a parallel viability assay (e.g., MTT or Live/Dead staining) to ensure observed effects are not due to cell death.

4. Vendors for Reagents and Antibodies

• Fluorescent Antibodies: BioLegend, BD Biosciences, Thermo Fisher Scientific.
• Fixation/Permeabilization Kits: BD Cytofix/Cytoperm, Invitrogen.
• Viability Dyes: Zombie Aqua™ (BioLegend), Live/Dead™ Fixable Dyes (Invitrogen).

 

 

Here is a Materials and Methods section for a Nitric Oxide (NO) Production Assay using the Griess Assay with monocytes and macrophages. This includes co-incubation of a test compound to trigger an immune reaction and measurement of nitric oxide production.

Materials and Methods

1. Materials

1.1 Cells and Culture Media

• Monocytes and Macrophages:
• Isolated from fresh human blood as described previously or purchased (e.g., Lonza, Stemcell Technologies, or BioIVT).
• Monocytes can be differentiated into macrophages by culturing with M-CSF (50 ng/mL) for 5–7 days.
• Culture Media:
• RPMI 1640 or DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% Penicillin-Streptomycin, and 2 mM L-glutamine.

1.2 Stimulants and Test Compound

• Test Compound:
• Pharmaceutical or other compound dissolved in an appropriate solvent (e.g., DMSO or PBS).
• Controls:
• Positive Control: Lipopolysaccharide (LPS, 1 µg/mL, Sigma-Aldrich) to induce nitric oxide production.
• Negative Control: Vehicle control (e.g., 0.1% DMSO or PBS).

1.3 Reagents for the Griess Assay

• Griess Reagents:
• Sulfanilamide (1% w/v in 5% phosphoric acid).
• N-(1-Naphthyl)ethylenediamine dihydrochloride (NED, 0.1% w/v in water).
• Sodium Nitrite (NaNO₂):
• For preparing a nitrite standard curve.
• Phosphate-Buffered Saline (PBS):
• Without calcium and magnesium.

1.4 Plasticware and Consumables

• 96-well flat-bottom tissue culture plates.
• 96-well flat-bottom ELISA plates (for Griess assay).
• Sterile pipette tips.
• 15 mL and 50 mL centrifuge tubes.

1.5 Equipment

• CO₂ incubator (37°C, 5% CO₂).
• Plate reader (compatible with 540 nm wavelength).

2. Methods

2.1 Cell Preparation

1. Monocyte Isolation and Macrophage Differentiation:

• Isolate monocytes or purchase cryopreserved cells.
• If macrophages are required, differentiate monocytes by plating them in RPMI 1640 with 10% FBS and M-CSF (50 ng/mL) for 5–7 days.

1. Plating Cells:

• Seed monocytes/macrophages into 96-well plates at a density of 1 × 10⁵ cells/well in 200 µL of culture medium.
• Allow macrophages to adhere overnight.

2.2 Co-Incubation with Test Compound

1. Preparation of Test Compound:

• Prepare serial dilutions of the test compound in culture media (e.g., 1 nM to 100 µM).
• Include controls:
• Positive Control: LPS (1 µg/mL).
• Negative Control: Vehicle (e.g., 0.1% DMSO or PBS).

1. Treatment:

• Replace culture media in each well with media containing the test compound or controls.
• Incubate cells at 37°C for 24 hours.

2.3 Griess Assay for Nitric Oxide Detection

1. Supernatant Collection:

• After 24 hours, carefully collect 100 µL of cell culture supernatant from each well and transfer to a fresh 96-well flat-bottom ELISA plate.
• Store supernatants at -80°C if not analyzing immediately.

1. Preparation of Griess Reagents:

• Prepare Sulfanilamide Solution (1% w/v in 5% phosphoric acid).
• Prepare NED Solution (0.1% w/v in distilled water).
• Mix equal volumes of the two solutions just before use.

1. Standard Curve Preparation:

• Prepare a sodium nitrite (NaNO₂) stock solution (1 mM in water).
• Perform serial dilutions in culture media to create a standard curve ranging from 0 to 50 µM nitrite.

1. Performing the Assay:

• Add 50 µL of the collected supernatant to each well of the ELISA plate.
• Add 50 µL of the freshly prepared Griess reagent to each well.
• Incubate the plate for 10–15 minutes at room temperature in the dark.

1. Reading the Plate:

• Measure the absorbance at 540 nm using a plate reader.

2.4 Data Analysis

1. Standard Curve:

• Plot the nitrite standard concentrations (x-axis) against the absorbance at 540 nm (y-axis) to generate a standard curve.
• Use the standard curve to calculate nitrite concentrations in the test samples.

1. Normalization:

• Normalize nitrite levels to the number of cells per well.

1. Statistical Analysis:

• Perform statistical tests (e.g., ANOVA or t-tests) to compare nitrite levels between treatment groups and controls.

3. Notes and Considerations

1. Solvent Compatibility:

• Ensure the solvent for the test compound (e.g., DMSO) does not exceed 0.1%, as higher concentrations may affect cell viability and NO production.

1. Cell Viability:

• Conduct a parallel viability assay (e.g., MTT, Trypan Blue exclusion) to confirm that observed effects are not due to cytotoxicity.

1. Low Nitric Oxide Producers:

• Human macrophages may not produce high levels of nitric oxide compared to mouse macrophages. Consider using THP-1 cells (human monocyte-derived macrophage-like cell line) if necessary.

1. Timepoint Optimization:

• While 24 hours is common, consider testing earlier or later time points (e.g., 6, 12, 48 hours) to capture the peak NO response.

4. Vendors for Reagents

• Griess Reagents: Sigma-Aldrich, Thermo Fisher Scientific.
• Nitrite Standards: Sigma-Aldrich.
• Cell Culture Media and Supplements: Gibco (Thermo Fisher Scientific).
• Macrophage Differentiation Reagents: PeproTech, BioLegend.

 

 

Here is a detailed Materials and Methods section for a Reactive Oxygen Species (ROS) Assay using monocytes and macrophages, including the co-incubation of a test compound to trigger an immune reaction.

Materials and Methods

1. Materials

1.1 Cells and Culture Media

• Monocytes and Macrophages:
• Isolated from fresh human blood or purchased from suppliers like Lonza, Stemcell Technologies, or BioIVT.
• For macrophages, monocytes are differentiated using M-CSF (50 ng/mL) for 5–7 days in RPMI 1640 or DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS).
• Culture Media:
• RPMI 1640 or DMEM, supplemented with:
• 10% heat-inactivated FBS.
• 1% Penicillin-Streptomycin.
• 2 mM L-glutamine.

1.2 ROS Detection Reagents

• Fluorescent ROS Probe:
• 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA, Thermo Fisher Scientific, Sigma-Aldrich, or similar).
• Cell-permeable probe that fluoresces when oxidized.
• Positive Control:
• Phorbol 12-myristate 13-acetate (PMA, 10 ng/mL, Sigma-Aldrich) to induce ROS.
• Negative Control:
• Vehicle control (e.g., 0.1% DMSO or PBS).

1.3 Additional Reagents

• Test Compound:
• Pharmaceutical or other compound dissolved in an appropriate solvent (e.g., DMSO or PBS).
• Phosphate-Buffered Saline (PBS):
• Without calcium and magnesium.
• Antioxidant Control (Optional):
• N-acetylcysteine (NAC, Sigma-Aldrich) as an inhibitor of ROS production.

1.4 Plasticware and Consumables

• Sterile 96-well black flat-bottom plates (preferred for fluorescence assays).
• 15 mL and 50 mL centrifuge tubes.
• Sterile pipette tips.

1.5 Equipment

• Fluorescence plate reader (compatible with excitation at 485 nm and emission at 535 nm).
• CO₂ incubator (37°C, 5% CO₂).

2. Methods

2.1 Cell Preparation

1. Monocyte Isolation and Macrophage Differentiation:

• Isolate monocytes or use cryopreserved monocytes.
• To differentiate into macrophages, plate monocytes at 1 × 10⁶ cells/mL in RPMI 1640 or DMEM supplemented with 10% FBS and M-CSF (50 ng/mL). Incubate for 5–7 days, replacing the medium every 2–3 days.

1. Plating Cells for Assay:

• Seed monocytes/macrophages into a 96-well black flat-bottom plate at a density of 1 × 10⁵ cells/well in 200 µL of complete media.
• Allow macrophages to adhere overnight.

2.2 Co-Incubation with Test Compound

1. Preparation of Test Compound:

• Prepare serial dilutions of the test compound in culture media (e.g., 1 nM to 100 µM).
• Include controls:
• Positive Control: PMA (10 ng/mL).
• Negative Control: Vehicle (e.g., 0.1% DMSO or PBS).
• Antioxidant Control (Optional): Add NAC (5 mM) to inhibit ROS.

1. Treatment:

• Replace culture media with media containing the test compound or controls.
• Incubate cells for 1–6 hours at 37°C and 5% CO₂, depending on the expected kinetics of ROS production.

2.3 ROS Detection with H₂DCFDA

1. Loading the ROS Probe:

• Prepare a working solution of H₂DCFDA (final concentration: 10 µM) in serum-free RPMI 1640 or DMEM.
• Remove media from each well and wash cells with PBS.
• Add 100 µL of H₂DCFDA solution to each well.
• Incubate at 37°C for 30–60 minutes in the dark.

1. Removing Excess Probe:

• After incubation, remove the H₂DCFDA solution and wash cells twice with PBS to remove excess probe.

1. Triggering ROS Production:

• Add 100 µL of media containing the test compound, positive control (PMA), or negative control to each well.
• Incubate at 37°C for 30 minutes to 1 hour in the dark.

2.4 Measurement of ROS

1. Fluorescence Measurement:

• Measure fluorescence using a plate reader:
• Excitation wavelength: 485 nm.
• Emission wavelength: 535 nm.
• Record fluorescence at multiple time points (e.g., 0, 15, 30, 60 minutes) to capture ROS dynamics.

1. Normalization:

• Normalize fluorescence intensity to the number of cells per well.

2.5 Data Analysis

1. Standard Curve (Optional):

• Prepare a standard curve using known concentrations of hydrogen peroxide (H₂O₂) to quantify ROS production in terms of H₂O₂ equivalents.

1. Statistical Analysis:

• Compare fluorescence intensity between test compound, positive control, and negative control groups using statistical tests (e.g., ANOVA or t-tests).

3. Notes and Considerations

1. Probe Sensitivity:

• H₂DCFDA is sensitive to light and can auto-oxidize. Prepare and handle solutions in low-light conditions.

1. Kinetics of ROS Production:

• ROS production may vary depending on the test compound. Perform preliminary time-course experiments to determine the optimal incubation time.

1. Cell Viability:

• Conduct a parallel viability assay (e.g., MTT or Trypan Blue exclusion) to ensure that changes in ROS are not due to cytotoxicity.

1. Controls for Non-Specific Oxidation:

• Include wells without cells to account for non-specific probe oxidation.

1. Additional Probes (Optional):

• Consider alternative ROS probes for specific ROS species:
• DHE (Dihydroethidium): Superoxide detection.
• MitoSOX™: Mitochondrial superoxide detection.

4. Vendors for Reagents

• H₂DCFDA: Thermo Fisher Scientific, Sigma-Aldrich.
• PMA: Sigma-Aldrich.
• NAC: Sigma-Aldrich, Millipore.
• Cell Culture Media and Supplements: Gibco (Thermo Fisher Scientific).

 

Here’s a detailed Materials and Methods section for conducting cell viability and proliferation assays with monocytes and macrophages. The method involves co-incubating cells with a test compound to trigger an immune reaction and assess the impact on cell viability and proliferation.

Materials and Methods

1. Materials

1.1 Cells and Culture Media

• Monocytes and Macrophages:
• Isolated from fresh human blood or purchased from Lonza, Stemcell Technologies, or BioIVT.
• If macrophages are needed, differentiate monocytes using M-CSF (50 ng/mL) in RPMI 1640 or DMEM supplemented with:
• 10% heat-inactivated fetal bovine serum (FBS).
• 1% Penicillin-Streptomycin.
• 2 mM L-glutamine.
• Culture Media:
• RPMI 1640 or DMEM, as above.

1.2 Cell Viability and Proliferation Assays

1. Cell Viability:

• MTT Assay:
• MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Sigma-Aldrich).
• Dimethyl sulfoxide (DMSO).
• Alternative Reagents:
• AlamarBlue™ (resazurin-based, Invitrogen).
• CellTiter-Glo™ (ATP-based, Promega).

1. Cell Proliferation:

• BrdU Assay:
• Bromodeoxyuridine (BrdU) incorporation assay kit (e.g., Roche or Sigma-Aldrich).
• Alternative Proliferation Assay:
• CFSE (Carboxyfluorescein Succinimidyl Ester, Invitrogen).

1.3 Stimulants and Test Compound

• Test Compound:
• Pharmaceutical or other compound, dissolved in DMSO or PBS.
• Controls:
• Positive Control: Lipopolysaccharide (LPS, 1 µg/mL) or phorbol 12-myristate 13-acetate (PMA, 10 ng/mL).
• Negative Control: Vehicle control (e.g., 0.1% DMSO or PBS).

1.4 Additional Reagents

• Phosphate-Buffered Saline (PBS): Without calcium and magnesium.
• Trypan Blue Solution (0.4%): For manual viability counting.

1.5 Plasticware and Consumables

• Sterile 96-well flat-bottom plates (for assays).
• 6-well plates (for cell culture).
• Pipette tips, sterile tubes, and cell scrapers.

1.6 Equipment

• CO₂ incubator (37°C, 5% CO₂).
• Microplate reader (compatible with 570 nm and 600 nm for MTT).
• Fluorescence or luminescence plate reader (for AlamarBlue™ or CellTiter-Glo™).
• Inverted microscope for cell visualization.

2. Methods

2.1 Cell Preparation

1. Monocyte Isolation and Macrophage Differentiation:

• Isolate monocytes or use cryopreserved cells.
• Differentiate into macrophages by culturing monocytes at 1 × 10⁶ cells/mL in RPMI 1640 or DMEM supplemented with M-CSF (50 ng/mL) for 5–7 days.

1. Plating for Assays:

• Seed monocytes/macrophages in a 96-well plate at 1 × 10⁵ cells/well in 200 µL of complete media.
• Allow macrophages to adhere overnight before treatment.

2.2 Co-Incubation with Test Compound

1. Preparation of Test Compound:

• Prepare serial dilutions of the test compound in culture media (e.g., 1 nM to 100 µM).
• Include the following controls:
• Positive Control: LPS or PMA to stimulate immune activation.
• Negative Control: Vehicle (e.g., 0.1% DMSO or PBS).

1. Treatment:

• Replace media in each well with media containing the test compound or controls.
• Incubate for 24–48 hours at 37°C in 5% CO₂.

2.3 Cell Viability Assays

MTT Assay

1. Adding MTT Reagent:

• Add 20 µL of MTT solution (5 mg/mL in PBS) to each well.
• Incubate at 37°C for 3–4 hours until formazan crystals form.

1. Dissolving Crystals:

• Carefully remove the supernatant without disturbing the crystals.
• Add 100 µL of DMSO to each well to dissolve the formazan crystals.

1. Measurement:

• Measure absorbance at 570 nm with a reference wavelength of 600 nm using a microplate reader.

AlamarBlue™ Assay (Optional)

1. Adding AlamarBlue™:

• Add 10 µL of AlamarBlue™ reagent directly to the culture media in each well.
• Incubate at 37°C for 1–4 hours.

1. Measurement:

• Measure fluorescence using a plate reader:
• Excitation: 560 nm.
• Emission: 590 nm.

CellTiter-Glo™ Assay (Optional)

1. Adding Reagent:

• Add an equal volume of CellTiter-Glo™ reagent (100 µL) to the media.
• Mix gently and incubate for 10 minutes at room temperature.

1. Measurement:

• Measure luminescence using a luminescence plate reader.

2.4 Cell Proliferation Assays

BrdU Assay

1. Labeling Cells with BrdU:

• Add 10 µL of BrdU labeling reagent to each well.
• Incubate for 4–24 hours, depending on the expected proliferation rate.

1. Fixation and Detection:

• Fix cells with the kit-provided fixation solution.
• Add anti-BrdU antibody and incubate as per the manufacturer’s protocol.

1. Measurement:

• Add substrate solution and measure absorbance at 370 nm using a plate reader.

CFSE Proliferation Assay

1. Labeling Cells with CFSE:

• Resuspend monocytes/macrophages in PBS containing 5 µM CFSE.
• Incubate at 37°C for 10 minutes in the dark.
• Wash cells twice with complete media to remove excess CFSE.

1. Plating and Treatment:

• Plate CFSE-labeled cells in a 96-well plate and treat with the test compound as described above.

1. Flow Cytometry Analysis:

• Harvest cells after the treatment period.
• Analyze CFSE fluorescence using a flow cytometer to assess proliferation (fluorescence dilution indicates proliferation).

2.5 Data Analysis

1. Normalization:

• Normalize viability/proliferation values to untreated controls.
• For MTT, calculate the percentage of viable cells using: Cell Viability (%)=(ODtestODcontrol)×100\text{Cell Viability (\%)} = \left( \frac{\text{OD}_{\text{test}}}{\text{OD}_{\text{control}}} \right) \times 100

1. Statistical Analysis:

• Compare test compound effects with controls using ANOVA or t-tests.

3. Notes and Considerations

1. Optimization:

• Perform preliminary experiments to determine optimal test compound concentrations and incubation times.

1. Controls:

• Include wells with no cells to account for background signal.

1. Cytotoxicity:

• For cytotoxic compounds, use shorter incubation times (e.g., 4–6 hours) to avoid confounding effects from extensive cell death.

1. Additional Validation:

• Combine viability assays (e.g., MTT) with apoptosis/necrosis markers (e.g., Annexin V/PI) for comprehensive evaluation.

4. Vendors for Reagents

• MTT Reagent: Sigma-Aldrich, Thermo Fisher Scientific.
• AlamarBlue™: Invitrogen (Thermo Fisher Scientific).
• BrdU Kits: Sigma-Aldrich, Roche.
• CellTiter-Glo™: Promega.
• CFSE: Invitrogen (Thermo Fisher Scientific).
• LPS, PMA: Sigma-Aldrich.

 

Here is a detailed Materials and Methods section for Gene Expression Profiling using qRT-PCR or RNA-Seq with monocytes and macrophages. This includes co-incubation with a test compound to trigger an immune reaction and measurement of changes in gene expression.

Materials and Methods

1. Materials

1.1 Cells and Culture Media

• Monocytes and Macrophages:
• Isolated from fresh human blood or purchased from Lonza, Stemcell Technologies, or BioIVT.
• If macrophages are needed, differentiate monocytes by culturing in RPMI 1640 or DMEM supplemented with:
• M-CSF (50 ng/mL) for 5–7 days.
• 10% heat-inactivated fetal bovine serum (FBS).
• 1% Penicillin-Streptomycin.
• 2 mM L-glutamine.
• Culture Media:
• RPMI 1640 or DMEM as above.

1.2 Stimulants and Test Compound

• Test Compound:
• Pharmaceutical or other compound dissolved in DMSO or PBS.
• Controls:
• Positive Control: Lipopolysaccharide (LPS, 1 µg/mL) to trigger an immune response.
• Negative Control: Vehicle control (e.g., 0.1% DMSO or PBS).

1.3 RNA Isolation and cDNA Synthesis

• RNA Isolation Kit:
• TRIzol™ Reagent (Thermo Fisher Scientific) or RNeasy Kit (Qiagen).
• DNase Treatment:
• DNase I (e.g., Qiagen RNase-Free DNase Set).
• cDNA Synthesis Kit:
• High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) or equivalent.

1.4 qRT-PCR Reagents

• qRT-PCR Master Mix:
• SYBR Green Master Mix (Thermo Fisher Scientific) or TaqMan™ Fast Advanced Master Mix.
• Primers:
• Custom primers for target genes and housekeeping genes (e.g., GAPDH, ACTB).
• PCR Plates:
• 96-well or 384-well PCR plates.

1.5 RNA-Seq Library Preparation (Optional)

• RNA-Seq Library Kit:
• NEBNext Ultra II RNA Library Prep Kit (New England Biolabs) or TruSeq Stranded Total RNA Library Prep Kit (Illumina).
• RNA Quality Control:
• Bioanalyzer (Agilent) or TapeStation for RNA integrity number (RIN) assessment.

1.6 Plasticware and Consumables

• Sterile 6-well plates for cell culture.
• RNase-free tubes and pipette tips.
• PCR tubes or plates.

1.7 Equipment

• CO₂ incubator (37°C, 5% CO₂).
• Microcentrifuge.
• Real-time PCR machine (e.g., QuantStudio™, Bio-Rad CFX96).
• Sequencer (e.g., Illumina NovaSeq, HiSeq, or MiSeq).

2. Methods

2.1 Cell Preparation

1. Monocyte Isolation and Macrophage Differentiation:

• Isolate monocytes from PBMCs or purchase cryopreserved cells.
• For macrophages, culture monocytes in RPMI 1640 or DMEM with M-CSF (50 ng/mL) for 5–7 days, refreshing media every 2–3 days.

1. Plating for Treatment:

• Plate monocytes or macrophages in 6-well plates at 1 × 10⁶ cells/well in 2 mL of complete media.
• Allow macrophages to adhere overnight.

2.2 Co-Incubation with Test Compound

1. Preparation of Test Compound:

• Prepare serial dilutions of the test compound in culture media (e.g., 1 nM to 100 µM).
• Include controls:
• Positive Control: LPS (1 µg/mL).
• Negative Control: Vehicle.

1. Treatment:

• Replace media with 2 mL of fresh media containing the test compound or controls.
• Incubate cells at 37°C for 4–24 hours, depending on the expected gene expression changes.

2.3 RNA Isolation

1. Harvesting Cells:

• After treatment, wash cells with ice-cold PBS.
• Lyse cells directly in the well using TRIzol™ Reagent (1 mL per well) or buffer from the RNeasy Kit.
• Transfer lysates to RNase-free tubes.

1. RNA Extraction:

• Extract RNA using the TRIzol protocol or RNeasy Kit as per the manufacturer’s instructions.
• Include DNase treatment to remove genomic DNA contamination.
• Quantify RNA using a spectrophotometer (e.g., NanoDrop) and assess purity (A260/A280 ratio).

1. RNA Quality Control:

• Check RNA integrity using a Bioanalyzer or TapeStation. Only samples with RIN >7 are suitable for RNA-Seq.

2.4 cDNA Synthesis

1. Reverse Transcription:

• Synthesize cDNA from 1 µg of RNA using a cDNA synthesis kit.
• Use random primers or oligo(dT) primers as specified in the kit.

2.5 Gene Expression Analysis

qRT-PCR

1. Primer Design:

• Design primers for target genes (e.g., pro-inflammatory cytokines: IL-6, TNF-α, IL-1β).
• Include housekeeping genes (e.g., GAPDH, ACTB).

1. qRT-PCR Reaction Setup:

• Prepare a reaction mix containing:
• 10 µL SYBR Green or TaqMan Master Mix.
• 1 µL of forward and reverse primers (final concentration: 200–500 nM).
• 1 µL cDNA (diluted 1:10).
• 8 µL nuclease-free water.
• Pipette 20 µL reaction mix into each well of a 96-well PCR plate.

1. Thermal Cycling Conditions:

• Initial denaturation: 95°C for 2 minutes.
• 40 cycles of:
• Denaturation: 95°C for 15 seconds.
• Annealing/Extension: 60°C for 1 minute.

1. Data Analysis:

• Calculate relative expression levels using the ΔΔCt method.

RNA-Seq

1. Library Preparation:

• Use 1 µg of RNA to prepare sequencing libraries with an RNA-Seq library prep kit.
• Use poly(A) selection or rRNA depletion for RNA enrichment.

1. Sequencing:

• Sequence libraries on an Illumina platform with a target depth of 20–30 million reads per sample.

1. Data Analysis:

• Perform quality control using FastQC.
• Align reads to the human genome using STAR or HISAT2.
• Quantify gene expression using featureCounts or Salmon.
• Perform differential expression analysis with DESeq2 or edgeR.

2.6 Data Analysis

1. Normalization:

• For qRT-PCR, normalize target gene expression to housekeeping genes (ΔCt).
• For RNA-Seq, normalize gene expression to transcripts per million (TPM) or fragments per kilobase of transcript per million mapped reads (FPKM).

1. Statistical Analysis:

• Use t-tests or ANOVA to compare groups (qRT-PCR).
• For RNA-Seq, apply statistical significance thresholds (e.g., adjusted p-value <0.05, fold change >2).

3. Notes and Considerations

1. Timepoints:

• Perform a time-course experiment to identify optimal time points for gene expression changes.

1. Controls:

• Include technical replicates (qRT-PCR) and biological replicates (n ≥ 3).

1. RNA-Seq Depth:

• For targeted gene analysis, lower sequencing depth (e.g., 10M reads/sample) may suffice.

4. Vendors for Reagents

• TRIzol™ Reagent: Thermo Fisher Scientific.
• qRT-PCR Master Mix: Bio-Rad, Thermo Fisher Scientific.
• RNA-Seq Library Prep Kits: Illumina, NEB.
• LPS, PMA: Sigma-Aldrich.

Here is a comprehensive Materials and Methods section for performing Western Blot (Immunoblotting) with monocytes and macrophages, including co-incubation with a test compound to trigger an immune reaction and the analysis of protein expression.

Materials and Methods

1. Materials

1.1 Cells and Culture Media

• Monocytes and Macrophages:
• Isolated from human blood or purchased from Lonza, Stemcell Technologies, or BioIVT.
• Macrophages differentiated by culturing monocytes in RPMI 1640 or DMEM supplemented with:
• M-CSF (50 ng/mL) for 5–7 days.
• 10% heat-inactivated fetal bovine serum (FBS).
• 1% Penicillin-Streptomycin.
• 2 mM L-glutamine.

1.2 Stimulants and Test Compound

• Test Compound:
• Pharmaceutical or other compound dissolved in an appropriate solvent (e.g., DMSO or PBS).
• Controls:
• Positive Control: Lipopolysaccharide (LPS, 1 µg/mL) to stimulate an immune response.
• Negative Control: Vehicle control (e.g., 0.1% DMSO or PBS).

1.3 Protein Extraction and Quantification

• Lysis Buffer:
• RIPA buffer (Radioimmunoprecipitation assay buffer, Thermo Fisher Scientific or Sigma-Aldrich) with added:
• Protease inhibitor cocktail (e.g., Roche Complete™ or Thermo Fisher Halt™).
• Phosphatase inhibitors (if analyzing phosphorylation, e.g., sodium orthovanadate or β-glycerophosphate).
• Protein Quantification Kit:
• BCA Protein Assay Kit (Thermo Fisher Scientific) or Bradford Assay Kit (Bio-Rad).

1.4 SDS-PAGE and Western Blotting

• Gel Preparation:
• Precast SDS-PAGE gels (4–20% gradient, Bio-Rad or Invitrogen).
• Alternatively, prepare gels with:
• Separating gel (e.g., 10–12% polyacrylamide).
• Stacking gel (4% polyacrylamide).
• Running Buffer:
• Tris-Glycine-SDS running buffer.
• Transfer Buffer:
• Tris-Glycine buffer with 20% methanol (optional: 0.1% SDS for high-molecular-weight proteins).
• Membrane:
• Nitrocellulose or PVDF membrane (e.g., Amersham Hybond, Millipore).

1.5 Primary and Secondary Antibodies

• Primary Antibodies:
• Target proteins (e.g., cytokines like IL-6, TNF-α; signaling molecules like NF-κB, STAT3, or phospho-p38 MAPK).
• Housekeeping protein: GAPDH, β-Actin, or Tubulin.
• Secondary Antibodies:
• HRP-conjugated anti-rabbit or anti-mouse IgG (depending on the host of the primary antibody, e.g., Cell Signaling Technology or Thermo Fisher).

1.6 Detection Reagents

• Chemiluminescent Substrate:
• ECL substrate (e.g., SuperSignal™ West Pico/Femto, Thermo Fisher).
• Film or Imaging System:
• X-ray film (Kodak) or chemiluminescent imaging system (e.g., Bio-Rad ChemiDoc).

2. Methods

2.1 Cell Preparation

1. Monocyte Isolation and Macrophage Differentiation:

• Isolate monocytes as previously described or use cryopreserved monocytes.
• For macrophages, culture monocytes in RPMI 1640 with M-CSF (50 ng/mL) for 5–7 days.

1. Plating for Treatment:

• Plate monocytes or macrophages in 6-well plates at 1 × 10⁶ cells/well in 2 mL of complete media.
• Allow macrophages to adhere overnight.

2.2 Co-Incubation with Test Compound

1. Preparation of Test Compound:

• Prepare serial dilutions of the test compound in culture media (e.g., 1 nM to 100 µM).
• Include controls:
• Positive Control: LPS (1 µg/mL).
• Negative Control: Vehicle (e.g., 0.1% DMSO or PBS).

1. Treatment:

• Replace media with 2 mL of fresh media containing the test compound or controls.
• Incubate for 4–24 hours depending on the target protein's expected response.

2.3 Protein Extraction

1. Harvesting Cells:

• After treatment, wash cells with ice-cold PBS.
• Add RIPA buffer (200–300 µL per well) supplemented with protease and phosphatase inhibitors.
• Scrape cells gently and transfer lysates to a pre-chilled microcentrifuge tube.

1. Lysate Preparation:

• Incubate lysates on ice for 30 minutes, vortexing every 5 minutes.
• Centrifuge at 14,000 x g for 15 minutes at 4°C to pellet debris.
• Transfer the supernatant (protein extract) to a fresh, pre-chilled tube.

1. Protein Quantification:

• Quantify protein concentration using the BCA Protein Assay Kit or Bradford Assay.

2.4 SDS-PAGE and Transfer

1. Loading Gel:

• Mix protein lysates with 4× Laemmli buffer (1:3 ratio) and heat at 95°C for 5 minutes.
• Load 20–30 µg of protein per well on a pre-cast or prepared SDS-PAGE gel.
• Run the gel at 120 V for 90 minutes or until the dye front exits.

1. Protein Transfer:

• Transfer proteins to a nitrocellulose or PVDF membrane using a wet or semi-dry transfer system:
• Wet Transfer: 100 V for 1 hour at 4°C.
• Semi-Dry Transfer: 25 V for 30 minutes.

2.5 Blocking and Antibody Incubation

1. Blocking:

• Block the membrane in 5% non-fat milk or 5% BSA in TBST (TBS + 0.1% Tween-20) for 1 hour at room temperature.

1. Primary Antibody Incubation:

• Incubate the membrane overnight at 4°C with the primary antibody diluted in TBST + 1% BSA (e.g., 1:1,000 dilution).

1. Secondary Antibody Incubation:

• Wash the membrane 3× with TBST for 5 minutes each.
• Incubate with HRP-conjugated secondary antibody (e.g., 1:5,000 dilution) in blocking buffer for 1 hour at room temperature.

2.6 Detection

1. Chemiluminescent Detection:

• Wash the membrane 3× with TBST for 5 minutes each.
• Add ECL substrate to cover the membrane and incubate for 1–2 minutes.

1. Imaging:

• Capture the signal using an X-ray film or a chemiluminescent imaging system.

2.7 Data Analysis

1. Densitometry:

• Quantify band intensities using ImageJ or software provided with the imaging system.
• Normalize target protein intensity to housekeeping protein intensity (e.g., GAPDH, β-Actin).

1. Statistical Analysis:

• Perform ANOVA or t-tests to compare treatments and controls.

3. Notes and Considerations

1. Optimization:

• Optimize antibody concentration and incubation times for specific proteins.
• Include loading controls (e.g., GAPDH or β-Actin).

1. Phosphorylation Studies:

• For phospho-specific antibodies, use freshly prepared lysis buffer with phosphatase inhibitors.

1. Low-Abundance Proteins:

• Use enhanced chemiluminescent substrates for detecting low-abundance proteins (e.g., SuperSignal™ West Femto).

4. Vendors for Reagents

• RIPA Buffer and Inhibitors: Thermo Fisher, Sigma-Aldrich.
• Gels and Buffers: Bio-Rad, Invitrogen.
• Antibodies: Cell Signaling Technology, Abcam, Thermo Fisher.
• ECL Substrates: Thermo Fisher (SuperSignal™), Bio-Rad (Clarity™).

 

 

Here is a detailed Materials and Methods section for conducting Macrophage Polarization Tests with monocytes and macrophages, including co-incubation with a test compound to trigger an immune reaction. This protocol assesses the polarization of macrophages into M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotypes and measures the associated markers.

Materials and Methods

1. Materials

1.1 Cells and Culture Media

• Monocytes and Macrophages:
• Isolated from fresh human blood or purchased from suppliers such as Lonza, Stemcell Technologies, or BioIVT.
• Macrophages differentiated by culturing monocytes in RPMI 1640 or DMEM supplemented with:
• M-CSF (50 ng/mL) for 5–7 days.
• 10% heat-inactivated fetal bovine serum (FBS).
• 1% Penicillin-Streptomycin.
• 2 mM L-glutamine.

1.2 Polarization Stimuli

• M1 Polarization Stimuli:
• Lipopolysaccharide (LPS, 1 µg/mL, Sigma-Aldrich).
• Recombinant human interferon-gamma (IFN-γ, 20 ng/mL, PeproTech or R&D Systems).
• M2 Polarization Stimuli:
• Recombinant human interleukin-4 (IL-4, 20 ng/mL) or interleukin-13 (IL-13, 20 ng/mL, PeproTech or R&D Systems).
• Test Compound:
• Pharmaceutical or other compound, dissolved in DMSO or PBS.

1.3 Reagents for Analysis

1. Cytokine Detection:

• Enzyme-linked immunosorbent assay (ELISA) kits for:
• IL-6, TNF-α, and IL-12 (M1 markers).
• IL-10, TGF-β, and Arg1 (M2 markers).

1. Flow Cytometry Reagents:

• Fluorescently conjugated antibodies for surface markers:
• M1 markers: CD80, CD86, HLA-DR.
• M2 markers: CD206 (mannose receptor), CD163, CD209.
• Intracellular antibodies for cytokines: IL-6, TNF-α, IL-10, TGF-β.

1.4 RNA and Protein Analysis Reagents (Optional)

• RNA isolation kit (e.g., TRIzol™ or RNeasy Kit).
• cDNA synthesis kit (e.g., Thermo Fisher High-Capacity cDNA Reverse Transcription Kit).
• qRT-PCR reagents (e.g., SYBR Green or TaqMan Master Mix).

1.5 Plasticware and Consumables

• Sterile 6-well plates for cell culture.
• 96-well plates for ELISA.
• RNase-free tubes and pipette tips.
• Flow cytometry tubes.

1.6 Equipment

• CO₂ incubator (37°C, 5% CO₂).
• Microplate reader for ELISA.
• Flow cytometer (e.g., BD FACSCanto II).
• Real-time PCR machine (e.g., Bio-Rad CFX96 or QuantStudio).

2. Methods

2.1 Cell Preparation

1. Monocyte Isolation and Macrophage Differentiation:

• Isolate monocytes from PBMCs or purchase cryopreserved cells.
• Differentiate into macrophages by plating monocytes at 1 × 10⁶ cells/mL in RPMI 1640 or DMEM with M-CSF (50 ng/mL) for 5–7 days. Replace the medium every 2–3 days.

1. Plating for Polarization:

• Plate macrophages in a 6-well plate at 1 × 10⁶ cells/well in 2 mL of complete medium.
• Allow macrophages to adhere overnight.

2.2 Polarization and Co-Incubation with Test Compound

1. Preparation of Test Compound:

• Prepare serial dilutions of the test compound in culture media (e.g., 1 nM to 100 µM).
• Include controls:
• Positive Controls: LPS + IFN-γ for M1 polarization, IL-4/IL-13 for M2 polarization.
• Negative Control: Vehicle (e.g., 0.1% DMSO or PBS).

1. Polarization and Treatment:

• Replace the media with 2 mL of fresh media containing the test compound, positive control stimuli, or vehicle control.
• Incubate for 24–48 hours at 37°C in 5% CO₂.

2.3 Endpoint Analyses

2.3.1 Cytokine Quantification (ELISA)

1. Supernatant Collection:

• Collect 100–200 µL of culture supernatant from each well.
• Centrifuge at 300 x g for 5 minutes to remove debris.
• Store at -80°C if not analyzing immediately.

1. ELISA:

• Perform ELISA for M1 and M2 cytokines (e.g., IL-6, TNF-α for M1; IL-10, TGF-β for M2) according to the manufacturer’s instructions.
• Measure absorbance at the appropriate wavelength using a plate reader (e.g., 450 nm).

2.3.2 Surface Marker Analysis (Flow Cytometry)

1. Cell Harvesting:

• Wash adherent cells with PBS and detach using cold PBS + EDTA (5 mM) or a cell scraper.
• Resuspend in FACS buffer (PBS + 2% FBS + 0.1% sodium azide).

1. Antibody Staining:

• For surface markers:
• Incubate cells with fluorescently labeled antibodies for CD80, CD86, HLA-DR (M1) or CD206, CD163, CD209 (M2) for 30 minutes at 4°C in the dark.
• For intracellular cytokines:
• Fix and permeabilize cells using an intracellular staining kit (e.g., BD Cytofix/Cytoperm).
• Incubate with cytokine antibodies (e.g., IL-6, TNF-α, IL-10, TGF-β) for 30 minutes at 4°C.

1. Flow Cytometry Analysis:

• Acquire at least 10,000 events per sample using a flow cytometer.
• Analyze using software such as FlowJo.

2.3.3 Gene Expression Analysis (qRT-PCR, Optional)

1. RNA Isolation:

• Extract total RNA from macrophages using TRIzol™ or RNeasy Kit.
• Quantify RNA and assess quality (A260/A280 ratio ≥1.8).

1. cDNA Synthesis:

• Reverse transcribe 1 µg of RNA using a cDNA synthesis kit.

1. qRT-PCR:

• Amplify target genes associated with M1 (e.g., IL-6, TNF-α, IL-12) or M2 (e.g., Arg1, CD206, TGF-β).
• Use GAPDH or ACTB as housekeeping genes.
• Perform relative quantification using the ΔΔCt method.

2.3.4 Protein Analysis (Western Blot, Optional)

• Analyze polarization-specific proteins (e.g., iNOS for M1; Arg1 for M2) by Western blot as described in a previous section.

2.4 Data Analysis

1. Normalization:

• Normalize ELISA and flow cytometry data to control groups.
• For qRT-PCR, calculate fold changes relative to housekeeping genes using ΔΔCt.

1. Statistical Analysis:

• Perform ANOVA or t-tests to compare test compound effects on M1/M2 polarization with controls.

3. Notes and Considerations

1. Kinetics:

• The timing of polarization and immune marker expression varies. Perform preliminary time-course experiments.

1. Cytotoxicity:

• Conduct a parallel cytotoxicity assay (e.g., MTT or Trypan Blue exclusion) to ensure the test compound is not inducing cell death.

1. Replicates:

• Include at least three biological replicates per condition for statistical robustness.

4. Vendors for Reagents

• Cytokines: PeproTech, R&D Systems.
• Antibodies: BioLegend, BD Biosciences, Thermo Fisher.
• RNA Isolation and qRT-PCR Kits: Qiagen, Thermo Fisher Scientific.
• ELISA Kits: Thermo Fisher Scientific, BD Biosciences.

 

 

Materials and Methods: Calcium Flux Assay with Monocytes and Macrophages

This protocol outlines the procedure for performing a calcium flux assay in monocytes and macrophages to assess intracellular calcium levels in response to co-incubation with a test compound, pharmaceutical, or immune stimulator.

1. Materials

1.1 Cells and Culture Media

• Monocytes and Macrophages:
• Isolated from human blood or purchased from Lonza, Stemcell Technologies, or BioIVT.
• Monocytes differentiated into macrophages by culturing in RPMI 1640 or DMEM supplemented with:
• M-CSF (50 ng/mL) for 5–7 days.
• 10% heat-inactivated fetal bovine serum (FBS).
• 1% Penicillin-Streptomycin.
• 2 mM L-glutamine.
• Calcium-Free Buffer:
• Hank's Balanced Salt Solution (HBSS) with:
• 1 mM MgCl₂.
• 1 mM CaCl₂.
• 20 mM HEPES (pH 7.4).

1.2 Calcium-Sensitive Dye

• Fluo-4 AM (Thermo Fisher Scientific) or similar fluorescent calcium indicator.
• Alternative dyes: Fura-2 AM (ratiometric), Indo-1 AM.
• Pluronic® F-127:
• Used to solubilize Fluo-4 AM.

1.3 Test Compound and Controls

• Test Compound:
• Pharmaceutical or other compound dissolved in DMSO or PBS.
• Controls:
• Positive Control: Ionomycin (calcium ionophore, 1 µM, Sigma-Aldrich) or ATP (100 µM, Sigma-Aldrich).
• Negative Control: Vehicle control (e.g., 0.1% DMSO or PBS).

1.4 Additional Reagents

• Quenching Dye (optional):
• Trypan Blue or similar dye to reduce extracellular fluorescence.
• Fixation Buffer:
• 4% paraformaldehyde (if cells need to be fixed for microscopy).

1.5 Plasticware and Consumables

• Black, clear-bottom 96-well plates (for fluorescence assays).
• Sterile pipette tips and centrifuge tubes.

1.6 Equipment

• Fluorescence plate reader (e.g., SpectraMax, BioTek) with appropriate filters:
• Excitation: 488 nm.
• Emission: 520 nm.
• CO₂ incubator (37°C, 5% CO₂).
• Flow cytometer (optional, for single-cell calcium flux analysis).

2. Methods

2.1 Cell Preparation

1. Monocyte Isolation and Macrophage Differentiation:

• Isolate monocytes or use cryopreserved monocytes.
• For macrophages, differentiate monocytes by plating at 1 × 10⁶ cells/mL in RPMI 1640 with M-CSF (50 ng/mL) for 5–7 days, replacing the medium every 2–3 days.

1. Plating Cells:

• Plate macrophages or monocytes in black, clear-bottom 96-well plates at 1 × 10⁵ cells/well in 200 µL of complete media.
• Allow macrophages to adhere overnight.

2.2 Co-Incubation with Test Compound

1. Preparation of Test Compound:

• Prepare serial dilutions of the test compound in calcium-free HBSS (e.g., 1 nM to 100 µM).
• Include controls:
• Positive Control: Ionomycin or ATP.
• Negative Control: Vehicle (e.g., 0.1% DMSO or PBS).

1. Treatment:

• Remove media from each well and replace with 200 µL of test compound or control solutions.
• Incubate cells at 37°C for 30 minutes before loading with the calcium-sensitive dye.

2.3 Dye Loading

1. Dye Preparation:

• Prepare a 5 µM working solution of Fluo-4 AM by diluting the stock solution in HBSS containing 0.02% Pluronic® F-127.
• Protect the dye from light.

1. Loading Dye into Cells:

• Remove the treatment solution and add 100 µL of the Fluo-4 AM solution to each well.
• Incubate cells at 37°C for 30–45 minutes in the dark.

1. Wash and Equilibration:

• Remove the dye solution and wash cells twice with calcium-free HBSS.
• Add 100 µL of HBSS containing 1 mM CaCl₂ and allow cells to equilibrate for 15 minutes at room temperature.

2.4 Measurement of Calcium Flux

1. Baseline Fluorescence:

• Measure baseline fluorescence at 488 nm excitation and 520 nm emission for 1–2 minutes to establish a baseline.

1. Test Compound Stimulation:

• Add 100 µL of test compound or controls to each well using a multi-channel pipette or automated dispenser while continuously recording fluorescence.

1. Dynamic Monitoring:

• Monitor changes in fluorescence for 3–10 minutes, depending on the expected kinetics of calcium flux.

2.5 Flow Cytometry (Optional)

1. Cell Harvesting:

• Detach macrophages using cold PBS and centrifuge at 300 x g for 5 minutes.
• Resuspend in HBSS with 1 mM CaCl₂ and Fluo-4 AM.

1. Acquisition:

• Analyze cells using a flow cytometer with a 488 nm laser for Fluo-4 excitation.
• Monitor real-time calcium flux over 2–5 minutes.

2.6 Data Analysis

1. Normalization:

• Normalize fluorescence intensity to baseline values (F/F₀) or maximum fluorescence for each condition.

1. Kinetics:

• Plot fluorescence intensity vs. time to observe dynamic changes in calcium levels.

1. Statistical Analysis:

• Use ANOVA or t-tests to compare test compound effects with positive and negative controls.

3. Notes and Considerations

1. Dye Sensitivity:

• Protect Fluo-4 AM from light to prevent degradation.
• Ensure cells are healthy, as compromised cells may leak dye or fail to respond.

1. Time Sensitivity:

• Perform calcium flux measurements immediately after dye loading to capture transient signals.

1. Positive Controls:

• Use ionomycin or ATP to confirm the responsiveness of cells in each experiment.

1. Kinetic Variability:

• Different compounds may elicit calcium flux at varying time points. Perform preliminary experiments to optimize time points for data acquisition.

4. Vendors for Reagents

• Fluo-4 AM: Thermo Fisher Scientific.
• Pluronic® F-127: Sigma-Aldrich.
• Ionomycin, ATP: Sigma-Aldrich or Thermo Fisher Scientific.
• Black, Clear-Bottom 96-Well Plates: Corning, Thermo Fisher.

 

Materials and Methods: Inflammasome Activation Assay

This protocol details an inflammasome activation assay using monocytes and macrophages to measure the immune reaction triggered by co-incubation with a test compound. Inflammasome activation typically involves the release of IL-1β and/or the cleavage of caspase-1.

1. Materials

1.1 Cells and Culture Media

• Monocytes and Macrophages:
• Freshly isolated human monocytes from PBMCs or cryopreserved monocytes (Lonza, Stemcell Technologies, BioIVT).
• Differentiation into macrophages using:
• M-CSF (50 ng/mL) in RPMI 1640 or DMEM supplemented with:
• 10% heat-inactivated fetal bovine serum (FBS).
• 1% Penicillin-Streptomycin.
• 2 mM L-glutamine.

1.2 Stimuli and Test Compound

• Test Compound:
• Pharmaceutical or other compound dissolved in DMSO or PBS.
• Priming Agent:
• Lipopolysaccharide (LPS, 1 µg/mL, Sigma-Aldrich) to upregulate inflammasome components (e.g., NLRP3 and pro-IL-1β).
• Inflammasome Activators:
• Nigericin (10 µM, Sigma-Aldrich) to activate NLRP3 inflammasome.
• ATP (5 mM, Sigma-Aldrich) as an alternative NLRP3 activator.

1.3 Reagents for Inflammasome Detection

• Cytokine Detection:
• IL-1β ELISA Kit (Thermo Fisher Scientific, BD Biosciences, or R&D Systems).
• Caspase-1 Activity Assay:
• FLICA™ Caspase-1 Assay Kit (ImmunoChemistry Technologies).
• Western Blot Reagents:
• Antibodies against cleaved IL-1β, cleaved caspase-1 (p20), and ASC (Abcam, Cell Signaling Technology).
• LDH Assay Kit (optional):
• For assessing cell death (Thermo Fisher Scientific).

1.4 RNA Analysis (Optional)

• RNA isolation kits (e.g., RNeasy Kit, Qiagen).
• cDNA synthesis kit (e.g., Thermo Fisher High-Capacity cDNA Kit).

1.5 Plasticware and Consumables

• 6-well plates for cell culture.
• 96-well plates for ELISA.
• Black 96-well plates for caspase-1 assays.
• Sterile pipette tips, RNase-free tubes.

1.6 Equipment

• CO₂ incubator (37°C, 5% CO₂).
• Microplate reader (compatible with fluorescence or absorbance detection).
• Flow cytometer (for caspase-1 activity assays).
• Western blotting equipment (for protein analysis).

2. Methods

2.1 Cell Preparation

1. Monocyte Isolation and Macrophage Differentiation:

• Isolate monocytes from PBMCs or use cryopreserved monocytes.
• Differentiate into macrophages by culturing monocytes at 1 × 10⁶ cells/mL in RPMI 1640 or DMEM with M-CSF (50 ng/mL) for 5–7 days. Replace media every 2–3 days.

1. Plating Cells:

• Seed monocytes or macrophages into 6-well plates at 1 × 10⁶ cells/well in 2 mL of complete media.
• Allow macrophages to adhere overnight.

2.2 Co-Incubation with Test Compound

1. Priming with LPS:

• Treat cells with LPS (1 µg/mL) for 3–4 hours at 37°C to induce expression of pro-IL-1β and NLRP3.

1. Inflammasome Activation:

• After LPS priming, treat cells with the test compound or inflammasome activators (e.g., Nigericin, ATP) for an additional 30 minutes to 2 hours:
• Test compound: serial dilutions (e.g., 1 nM to 100 µM).
• Positive Control: Nigericin (10 µM) or ATP (5 mM).
• Negative Control: Vehicle (e.g., 0.1% DMSO or PBS).

2.3 Endpoint Assays

2.3.1 IL-1β Detection (ELISA)

1. Supernatant Collection:

• Collect culture supernatants from each well and centrifuge at 300 x g for 5 minutes to remove debris.
• Store supernatants at -80°C if not analyzing immediately.

1. ELISA:

• Perform ELISA for IL-1β following the manufacturer's instructions.
• Measure absorbance at 450 nm using a plate reader.

2.3.2 Caspase-1 Activity Assay

1. FLICA Dye Loading:

• Add FLICA™ reagent directly to the culture wells at the recommended concentration.
• Incubate for 1 hour at 37°C in the dark.

1. Cell Harvesting:

• Detach adherent cells using PBS with 5 mM EDTA or a cell scraper.
• Wash cells twice with PBS.

1. Flow Cytometry:

• Analyze fluorescence intensity using a flow cytometer (excitation: 488 nm, emission: 520 nm) to detect active caspase-1.

2.3.3 Western Blot for Cleaved IL-1β and Caspase-1

1. Protein Extraction:

• Lyse cells using RIPA buffer supplemented with protease and phosphatase inhibitors.
• Centrifuge lysates at 14,000 x g for 15 minutes at 4°C and collect supernatants.

1. SDS-PAGE and Transfer:

• Load 20–30 µg of protein per well onto a 12% SDS-PAGE gel.
• Transfer proteins to a PVDF membrane.

1. Antibody Incubation:

• Probe with primary antibodies for cleaved IL-1β (17 kDa) and cleaved caspase-1 (p20).
• Incubate with HRP-conjugated secondary antibodies and detect using ECL substrate.

2.3.4 LDH Release Assay (Optional)

1. Supernatant Collection:

• Collect culture supernatants as described for ELISA.

1. LDH Assay:

• Perform LDH assay according to the manufacturer's protocol.
• Measure absorbance at 490 nm using a plate reader.

2.4 Data Analysis

1. Normalization:

• Normalize IL-1β or caspase-1 activity to untreated controls.

1. Statistical Analysis:

• Perform ANOVA or t-tests to compare test compound effects with positive and negative controls.

3. Notes and Considerations

1. Timing:

• Optimize the timing of priming and activation for specific test compounds.
• Typically, LPS priming requires 3–4 hours, and inflammasome activation occurs within 30 minutes to 2 hours.

1. Specificity:

• Include inhibitors of inflammasome activation (e.g., MCC950 for NLRP3) to confirm pathway specificity.

1. Replicates:

• Include at least three biological replicates for statistical robustness.

4. Vendors for Reagents

• LPS, Nigericin, ATP: Sigma-Aldrich, Thermo Fisher Scientific.
• IL-1β ELISA Kit: Thermo Fisher Scientific, BD Biosciences, R&D Systems.
• FLICA Caspase-1 Assay Kit: ImmunoChemistry Technologies.
• Antibodies for Western Blot: Cell Signaling Technology, Abcam.
• LDH Assay Kit: Thermo Fisher Scientific.

 

Materials and Methods: Chemotaxis Assay with Monocytes and Macrophages

This protocol describes a chemotaxis assay to measure the migration of monocytes and macrophages toward a chemoattractant, pharmaceutical compound, or another stimulus. The assay quantifies cell migration in response to chemokines, test compounds, or immune activators.

1. Materials

1.1 Cells and Culture Media

• Monocytes and Macrophages:
• Isolated from fresh human blood (e.g., via density gradient centrifugation) or purchased (Lonza, Stemcell Technologies, or BioIVT).
• For macrophages, differentiate monocytes in RPMI 1640 or DMEM with:
• M-CSF (50 ng/mL) for 5–7 days.
• 10% heat-inactivated fetal bovine serum (FBS).
• 1% Penicillin-Streptomycin.
• 2 mM L-glutamine.
• Chemotaxis Buffer:
• Serum-free RPMI 1640 or DMEM supplemented with 0.1% bovine serum albumin (BSA).

1.2 Test Compound and Controls

• Test Compound:
• Pharmaceutical or other compound dissolved in PBS or DMSO.
• Chemoattractants:
• Positive Control: CCL2 (MCP-1, 10–100 ng/mL, PeproTech or R&D Systems) for monocytes/macrophages.
• Negative Control: Vehicle (e.g., 0.1% DMSO or PBS).
• Blocking Agents (Optional):
• Chemokine receptor antagonists to confirm specificity (e.g., CCR2 inhibitor for MCP-1).

1.3 Chemotaxis Assay System

• Transwell Migration System:
• 24-well Transwell plates with 5 µm pore size inserts for monocytes or 8 µm for macrophages (e.g., Corning Costar Transwell System).
• Alternative Systems:
• µ-Slide Chemotaxis Chambers (ibidi) for imaging-based migration analysis.

1.4 Reagents for Analysis

• Cell Viability Dye:
• Trypan Blue or Live/Dead™ Stain (Invitrogen).
• Staining Reagents (Optional):
• DAPI, calcein-AM, or CFSE for labeling cells.
• Fixation Buffer:
• 4% paraformaldehyde (PFA) in PBS for fixed-cell assays.

1.5 Plasticware and Consumables

• Sterile 24-well plates.
• Sterile pipette tips.
• Centrifuge tubes.

1.6 Equipment

• CO₂ incubator (37°C, 5% CO₂).
• Inverted microscope (phase contrast or fluorescence).
• Hemocytometer or cell counter.

2. Methods

2.1 Cell Preparation

1. Monocyte Isolation and Macrophage Differentiation:

• Isolate monocytes using Ficoll-Paque or a similar density gradient method.
• Differentiate into macrophages by culturing monocytes at 1 × 10⁶ cells/mL in RPMI 1640 or DMEM supplemented with M-CSF (50 ng/mL) for 5–7 days.

1. Cell Harvesting and Counting:

• Detach macrophages using PBS with 5 mM EDTA or a cell scraper.
• Resuspend monocytes or macrophages in chemotaxis buffer at 1 × 10⁶ cells/mL.

2.2 Transwell Chemotaxis Assay

1. Preparation of the Transwell Plate:

• Add 600 µL of chemotaxis buffer containing the test compound, positive control (e.g., CCL2), or vehicle control to the lower chamber.
• Leave the upper chamber empty at this stage.

1. Loading Cells into the Transwell Insert:

• Add 100 µL of the cell suspension (1 × 10⁶ cells/mL) to the upper chamber of each Transwell insert.

1. Incubation:

• Incubate the Transwell plate at 37°C in 5% CO₂ for 3–6 hours (time may vary depending on cell type and chemoattractant).

1. Migration Analysis:

• After incubation, carefully remove the Transwell insert:
• For migrated cells:
• Collect the cells from the lower chamber.
• Stain cells with viability dye (e.g., Trypan Blue) or fluorescent markers for counting.
• For non-migrated cells:
• Optionally fix and stain cells remaining on the underside of the Transwell insert.

2.3 Quantification of Migrated Cells

1. Manual Counting:

• Use a hemocytometer to count live cells in the lower chamber after staining with Trypan Blue.

1. Fluorescence or Imaging-Based Counting (Optional):

• If cells are fluorescently labeled (e.g., calcein-AM or CFSE), count migrated cells using an inverted fluorescence microscope or a flow cytometer.

1. Automated Systems:

• Use imaging software or cell counters to quantify migrated cells from captured images.

2.4 Imaging-Based Chemotaxis Assay (ibidi µ-Slide)

1. Chamber Preparation:

• Load chemoattractant and test compound into one reservoir of the ibidi µ-Slide.
• Load cell suspension into the opposite reservoir.

1. Live Imaging:

• Place the slide in an incubated microscope stage and record images every 10 minutes for 3–6 hours.

1. Cell Tracking:

• Analyze migration paths and directionality using image analysis software (e.g., ImageJ with chemotaxis plugin).

2.5 Data Analysis

1. Migration Index:

• Calculate the percentage of migrated cells: Migration (%)=(Number of cells in lower chamberTotal number of cells)×100\text{Migration (\%)} = \left( \frac{\text{Number of cells in lower chamber}}{\text{Total number of cells}} \right) \times 100

1. Directional Migration (ibidi µ-Slide):

• Plot migration trajectories and calculate parameters such as velocity, directionality, and chemotactic index.

1. Statistical Analysis:

• Perform ANOVA or t-tests to compare migration rates between test compound, positive control, and vehicle control.

3. Notes and Considerations

1. Pore Size:

• Select pore sizes appropriate for your cell type:
• 5 µm for monocytes.
• 8 µm for macrophages.
• Larger pore sizes may allow non-specific migration.

1. Cell Health:

• Ensure cells are healthy and viable before the assay. Use a viability assay (e.g., Trypan Blue) to confirm >90% viability.

1. Optimization:

• Perform preliminary experiments to determine the optimal concentration of test compounds and incubation times.

1. Blocking Studies:

• Use receptor antagonists or neutralizing antibodies to confirm the specificity of chemotaxis toward the test compound.

4. Vendors for Reagents

• CCL2 (MCP-1), Test Compounds: PeproTech, R&D Systems.
• Transwell Inserts: Corning, Sigma-Aldrich.
• Fluorescent Labels (e.g., CFSE, calcein-AM): Thermo Fisher Scientific.
• ibidi µ-Slide Chemotaxis Chamber: ibidi GmbH.

Materials and Methods: Luminex Assays with Monocytes and Macrophages

This protocol describes the use of Luminex assays to measure multiple cytokines, chemokines, and other immune markers secreted by monocytes and macrophages in response to co-incubation with a test compound, pharmaceutical, or immune activator. The Luminex platform allows simultaneous quantification of multiple analytes in a single sample.

1. Materials

1.1 Cells and Culture Media

• Monocytes and Macrophages:
• Isolated from human blood or purchased from Lonza, Stemcell Technologies, or BioIVT.
• Macrophages differentiated by culturing monocytes in RPMI 1640 or DMEM supplemented with:
• M-CSF (50 ng/mL) for 5–7 days.
• 10% heat-inactivated fetal bovine serum (FBS).
• 1% Penicillin-Streptomycin.
• 2 mM L-glutamine.

1.2 Test Compounds and Controls

• Test Compound:
• Pharmaceutical or other compound dissolved in PBS or DMSO.
• Controls:
• Positive Control: Lipopolysaccharide (LPS, 1 µg/mL, Sigma-Aldrich) to trigger an immune response.
• Negative Control: Vehicle control (e.g., 0.1% DMSO or PBS).

1.3 Luminex Assay Kit

• Luminex Cytokine/Chemokine Assay Kit (e.g., Thermo Fisher Scientific, Bio-Rad, or R&D Systems):
• Multiplex beads pre-coated with capture antibodies for selected analytes (e.g., IL-6, TNF-α, IL-10, IL-1β, MCP-1, IFN-γ).
• Detection antibodies and streptavidin-phycoerythrin (SA-PE) reagent.
• Assay buffer, wash buffer, and standards.

1.4 Reagents for Sample Collection

• Phosphate-buffered saline (PBS) without calcium or magnesium.
• Sterile collection tubes (1.5 mL Eppendorf tubes).
• Protease inhibitors (optional, for stabilizing secreted proteins).

1.5 Consumables

• 96-well plate (non-binding, black or clear-bottom for Luminex).
• Pipette tips, filter tips, and centrifuge tubes.
• Parafilm or plate sealers.

1.6 Equipment

• CO₂ incubator (37°C, 5% CO₂).
• Centrifuge.
• Magnetic plate washer (for Luminex beads) or manual vacuum manifold.
• Luminex instrument (e.g., MAGPIX®, Luminex® 200™, Bio-Plex® 200).

2. Methods

2.1 Cell Preparation

1. Monocyte Isolation and Macrophage Differentiation:

• Isolate monocytes using Ficoll-Paque or purchase cryopreserved cells.
• For macrophages, differentiate monocytes by culturing at 1 × 10⁶ cells/mL in RPMI 1640 or DMEM with M-CSF (50 ng/mL) for 5–7 days. Replace media every 2–3 days.

1. Plating for Assay:

• Plate monocytes or macrophages in a 24-well plate at 5 × 10⁵ cells/well in 1 mL of complete media.
• Allow macrophages to adhere overnight.

2.2 Co-Incubation with Test Compound

1. Preparation of Test Compound:

• Prepare serial dilutions of the test compound in culture media (e.g., 1 nM to 100 µM).
• Include controls:
• Positive Control: LPS (1 µg/mL).
• Negative Control: Vehicle (e.g., 0.1% DMSO or PBS).

1. Treatment:

• Replace media with fresh media containing the test compound or controls.
• Incubate at 37°C for 24 hours in a CO₂ incubator.

2.3 Supernatant Collection

1. Harvesting Conditioned Media:

• After incubation, collect the culture supernatants (200–500 µL) from each well into labeled 1.5 mL tubes.
• Centrifuge at 300 x g for 5 minutes to remove debris.
• Aliquot supernatants and store at -80°C if not analyzing immediately.

2.4 Luminex Assay

1. Preparation of Standards:

• Reconstitute the lyophilized standard mix provided in the kit.
• Prepare a serial dilution series in assay buffer to generate a standard curve (e.g., 7 points: 5000 pg/mL to 1 pg/mL).

1. Bead Preparation:

• Resuspend magnetic or polystyrene beads coated with capture antibodies by vortexing.
• Add 50 µL of bead suspension to each well of a 96-well plate.

1. Sample Loading:

• Add 50 µL of standards (for standard curve) or 50 µL of samples (supernatants) to the appropriate wells.

1. Incubation:

• Seal the plate with parafilm or a plate sealer and incubate on a shaker at room temperature for 1–2 hours (or overnight at 4°C for increased sensitivity).

1. Washing:

• Wash beads 3× using the magnetic plate washer or manual vacuum manifold to remove unbound material.
• Use 200 µL of wash buffer per well for each wash.

1. Detection Antibody Binding:

• Add 50 µL of biotinylated detection antibody mix to each well.
• Incubate for 30 minutes at room temperature on a shaker.

1. Streptavidin-PE Binding:

• Add 50 µL of streptavidin-phycoerythrin (SA-PE) reagent to each well.
• Incubate for 30 minutes at room temperature in the dark.

1. Final Wash:

• Wash the beads 3× to remove excess SA-PE.
• Resuspend beads in 100 µL of assay buffer.

1. Data Acquisition:

• Read the plate on a Luminex instrument using recommended settings:
• Bead region classification.
• Reporter channel fluorescence (PE).
• Collect at least 50 events per bead region per well.

2.5 Data Analysis

1. Standard Curve:

• Generate a standard curve by plotting the known concentrations of standards (x-axis) against median fluorescence intensity (MFI) values (y-axis).
• Fit the curve using a 5-parameter logistic regression.

1. Quantification:

• Use the standard curve to calculate the concentration of each analyte in the supernatants.
• Normalize cytokine levels to cell number if required.

1. Statistical Analysis:

• Compare cytokine levels between test compound, positive control, and vehicle control groups using ANOVA or t-tests.

3. Notes and Considerations

1. Sample Dilution:

• Dilute samples if analyte concentrations exceed the upper limit of quantification.
• Perform pilot runs to determine the optimal sample dilution.

1. Multiplexing:

• Select analytes based on the expected immune response. Avoid overlapping analyte targets that may interfere with signal detection.

1. Replicates:

• Run samples and standards in duplicate or triplicate for statistical accuracy.

1. Bead Stability:

• Resuspend beads thoroughly before use to ensure consistent performance.

1. Instrument Calibration:

• Calibrate the Luminex instrument using the calibration and verification beads provided by the manufacturer.

4. Vendors for Reagents

• Luminex Assay Kits: Thermo Fisher Scientific (ProcartaPlex™), Bio-Rad (Bio-Plex™), MilliporeSigma (Milliplex™).
• Cytokines/Chemokines: PeproTech, R&D Systems.
• Cell Culture Reagents: Gibco (Thermo Fisher Scientific).

With the support of LLMs we are using gene data bases to complement our testings with AI:

Klar2O is TÜV SÜD certified & VDMA24364

 

Contact Us

To learn more about our testing process or to apply for the Makrolife Biotech Seal, please contact us:

Makrolife Biotech GmbH
Kirrlacherstr. 6
76646 Bruchsal, Germany
Email: info@makrolife-biotech.com
Website: www.makrolife-biotech.com