ICS

thinkpeptides is a brand of ProImmune Tel: +44 (0) 870 042 7279 — Fax: + 44 (0) 870 712 0588 — Email: peptides@thinkpeptides.com Web: www.thinkpeptides.com thinkpeptides — The Magdalen Centre Oxford Science Park — Oxford OX4 4GA — United Kingdom Registered in England No.: 387 3386 — VAT Registration No.: GB 749 9053 87 Intracellular Cytokine Staining Intracellular cytokine staining is a widely used flow cytometry based assay which detects the production and accumulation of cytokines within the endoplasmic reticulum. It can be used in combination with a variety of antibodies for cytokines and cellular markers, as well as MHC multimers for in depth phenotypic and functional analysis of a cell population. The following protocol has been optimised for use in human PBMCs. Experiments involving other cell types will require further modification. Materials Required • Anti-cytokine antibody conjugated to a fluorescent label (e.g. anti-Human IFNγ-FITC; ProImmune) • Anti-CD8 antibody • Peptide (stock at 50 mg/ml in DMSO - may be aliquotted and stored at -20ºC) • Leukocyte activation cocktail (LAC; optional) for use as a control for cytokine production (BD Biosciences #550583 - may be aliquotted and stored at -80ºC) • Cytokine IC control cells (optional) for use as a positive control when establishing the intracellular cytokine staining protocol (eBioscience #00-4509) • PBS Wash (2% fetal calf serum, 0.1% sodium azide in Phosphate buffered saline) • Permeabilization buffer (0.1% saponin, 1% fetal calf serum, 0.1% sodium azide in PBS) • 4% Paraformaldehyde • Fix solution (1% fetal calf serum, 2.5% formaldehyde in PBS) • Complete RPMI medium (500 ml RPMI-1640 supplemented with 5 ml Penicillin/Streptomycin/L-Glutamine (100x) and 37.5 ml fetal calf serum) • Brefeldin A (Sigma #15870) Protocol applicable for intracellular staining of IFNγγγγ, TNFαααα, MIP-1ββββ, or IL-2 All procedures up until step 7 should be carried out in a laminar flow hood, using sterile buffers and aseptic conditions. 1. Prepare peripheral blood cells in PBS Wash at a cell concentration of 2 × 107 cells/ml. 2. Transfer the cell suspension to individual tubes in 50 µl aliquots. 3. Add 500 µl PBS Wash to each tube. Centrifuge at 450 × g for 5 minutes at 10°C. 4. Aspirate supernatant. Agitate to disrupt cell pellets and resuspend in 200 µl complete RPMI. 5. Dilute peptide stock 1:50 in complete RPMI. Add 2 µl of this (10 µg/ml final*) to each desired tube. If using Leukocyte Activation cocktail (LAC) as a control, rapidly thaw this at 37°C in a water bath and add 0.33 µl of this to each desired tube. Note: After use, discard the unused portion of Leukocyte Activation cocktail. 6. Place the tubes at 37°C in a humidified CO2 incubator for 15 minutes to 1 hour. 7. Add Brefeldin A (10 µg/ml final) to the desired tubes (n.b. LAC contains Brefeldin A) and return to the incubator. Incubate for 15 hours†. 8. Remove tubes from the incubator. Centrifuge at 450 × g for 5 minutes at 10°C. 9. Aspirate supernatant. Resuspend desired cell pellets in 50 µl PBS Wash containing an optimally titrated amount§ of anti-CD8 antibody. Add 50 µl PBS Wash to remaining tubes. Note: Single-color controls should be stained at this stage. If additional phenotyping of samples is desired, antibodies to other cell surface receptors may also be added at this time. 10. Incubate for 20 minutes on ice. 11. Add 500 µl PBS Wash to each tube. Centrifuge at 450 × g for 5 minutes at 10°C. 12. Aspirate supernatant. Agitate to disrupt cell pellets. Protocol for Intracellular Cytokine Staining in Human PBMCs thinkpeptides is a brand of ProImmune Tel: +44 (0) 870 042 7279 — Fax: + 44 (0) 870 712 0588 — Email: peptides@thinkpeptides.com Web: www.thinkpeptides.com thinkpeptides — The Magdalen Centre Oxford Science Park — Oxford OX4 4GA — United Kingdom Registered in England No.: 387 3386 — VAT Registration No.: GB 749 9053 87 13. Add 200 µl 4% paraformaldehyde to each sample tube. Vortex tubes. Incubate for 20 minutes on ice. This step will fix the cell morphology of the activated cells. Note: The procedure can be stopped at this point. Repeat steps 12 and 13. Resuspend the cells in 100 µl/tube PBS Wash. Cover and store the cells at 4°C for up to 3 days. To proceed, repeat steps 12 and 13. Resuspend the cells in 200 µl/tube permeabilization buffer and proceed to step 16. 14. Add 200 µl permeabilization buffer to each tube. 15. Centrifuge at 450 × g for 5 minutes at 10°C. Aspirate supernatant. 16. Add 100 µl permeabilization buffer to the sample tubes that are to be stained with anti-cytokine antibody. Add 100 µl PBS Wash to the remaining tubes (i.e. Single-color controls). 17. Incubate for 5 minutes at room temperature. 18. Add an optimally titrated amount§ of FITC-conjugated anti-cytokine antibody to the desired sample tubes and mix. 19. Incubate for 20 minutes at room temperature. 20. Add 200 µl permeabilization buffer to each tube and centrifuge at 450 × g for 5 minutes at 10°C. Aspirate supernatant and agitate tubes to disrupt the cell pellets. 21. Resuspend the cells in 200 µl fix solution. Vortex tubes. It is important to vortex well when adding this fixative so that cells do not clump. 22. The samples are now ready for data acquisition and analysis on a flow cytometer but may be stored overnight at 4°C in the dark prior to analysis. Protocol Optimization † Incubation period The optimal stimulation period for induction of a given cytokine is variable and has to be determined. The incubation period of 16 hours described above was determined to be optimal for human PBMCs stimulated with 10 µg/ml peptide to induce an IFNγ response. The incubation period will vary dependent on the method of stimulation and the cytokine response to be determined. * Peptide concentration Investigate the effect on cytokine response of titrating the peptide. § Antibody concentrations Investigate the effect of titrating the anti-CD8 and anti-cytokine antibodies Protein Transport Inhibitor Brefeldin A is an inhibitor of intracellular protein transport. Incubation of cells in culture with Brefeldin A leads to blockade of protein transport to the Golgi complex and accumulation of proteins in the endoplasmic reticulum. Brefeldin A is effective for enhanced detection of a majority of intracellular cytokines; however, it is advised that you investigate the use and efficacy of this reagent as well as other protein transport inhibitors such as Monensin in your specific assay system. thinkpeptides is a brand of ProImmune  Copyright ProImmune Limited 2001-2008. All Rights Reserved. PR02TP Version 1.0 09/2008