M004 - Intracellular Cytokine Staining for Mouse Samples

Author Stephanie Swift Date 21st March 2017 Version 8 SOP # M004 Objective: This protocol addresses the processing, peptide-stimulation and antibody staining of fresh mouse samples for intracellular cytokine staining in 96-well U- bottom plates by flow cytometry. M004 - Intracellular Cytokine Staining for Mouse Samples Required reageants and equipment: ACK lysis buffer (see Appendix A) 1x Heparin (see Appendix A) (Sigma #H3149) Capped 5ml polystyrene tubes (BD Biosciences #352054) FACS Buffer (0.5% BSA (fraction V) in azide-free PBS) DC medium (see Appendix A) Golgi Plug (BD Biosciences #554715) Cytofix-Cytoperm (BD Biosciences #554715) Permwash buffer (BD Biosciences #554715; 10x) Fixable Viability Dye efluor450 (ebioscience #65-0863-18) CD8-Alexa Fluor 488 (Clone 53-6.7; BD Biosciences 557668) Optional: CD4-Brilliant Violet 510 (BioLegend #100449) Optional: CD3-Brilliant Violet 786 (BioLegend #100231) IFNg-Alexa Fluor 647 (Clone XMG-1.2; BD Biosciences #557735) TNFa-PE-Cy7 (Clone MP6-XT22; BD Biosciences #561041) IL-2-PE (Clone JES6-5H4; BD Biosciences #554428) 96-well U-bottom plates DMSO Phorbol 12-myristate 13-acetate (PMA) (Sigma #P-1585) Ionomycin (Sigma #I-0634) Test Peptides (See Appendix B) One Comp ebeads (ebioscience #01-1111-42) PBS without Ca/Mg A. Processing Blood Samples Blood samples are collected from a non-terminal saphenous bleed (~5 drops per mouse into a 5ml tube containing 200ul 1 x sterile heparin; see Appendix A). Always collect blood from two extra naive mice for your negative control (no peptide), positive control (PMA/ionomycin), unstained compensation control and your viability dye single stain control (see Appendix B, C). 1. Measure the total volume of blood collected for each individual mouse, and note this down in your labbook. This will allow you to convert frequencies into absolute numbers after running samples on the flow cytometer (see Appendix D). Remember to subtract the 200ul 1 x heparin collection solution. 2. Add 2ml ACK to each tube, flick to resuspend and incubate for 5 mins. at RT. 3. Wash with 2ml PBS. Spin down at 1500RPM for 5 mins. at 4 C. 4. Pour off supernatant, blot on tissue, and resuspend by flicking. 5. Add 2ml ACK to each tube, flick to resuspend and incubate for 5 mins. at RT. 6. Wash with 2ml PBS. Spin down at 1500RPM for 5 mins. at 4 C. 1

7. Pour off supernatant, blot on tissue, and resuspend by flicking. 8. Add 2ml DC medium to wash. Spin down at 1500RPM for 5 mins. at 4 C, remove supernatant, blot on tissue & resuspend by flicking. 9. Resuspend in a volume of DC medium that allows you to plate 100ul per well of a 96-well U-bottom plate, taking into account that there will be ~ 50ul left in the tube after decanting the final DC medium wash. N.B. If you don't have sufficient cells to attain this from a non-terminal bleed, resuspend in a final volume of DC medium that allows 50ul for unstained control samples and 100ul for experimental samples per well. B. Peptide Stimulation 10. Add 50ul/well of test peptide(s), diluted in DC medium, to each sample. For negative (unstimulated) controls, add 50ul/well of DMSO diluted in DC medium (see Appendix B). For positive controls and viability dye single stain controls, add 50ul/well of PMA/ionomycin (see Appendix B). N.B. For weak peptides, further stimulation may be necessary with anti- CD28 & anti-cd49d. 11. Incubate for 1h at 37 C. 12. Add 50ul diluted Golgi Plug to both unstimulated and stimulated wells. Incubate for a further 4h at 37 C. See below for example calculation for 20 wells:- (always make extra to account for pipetting loss/error) Reageant Dilution Volume (ul) Golgi Plug 0.2ul per well 4 DC medium - 996 13. Spin down at 1500RPM for 5 mins. at 4 C. - If you wish to leave cells overnight at this point, resuspend cells in 200ul DC medium. Store at 4 C overnight IN THE DARK. Proceed with step 14 the following day. - If you wish to continue immediately with staining, proceed with step 15. C. Intracellular Cytokine Staining (ICS) See Appendix C for information on preparing compensation controls 14. Spin down plate at 1500RPM for 5 mins. at 4 C and tip off supernatant, blotting on tissue. 15. Add 200ul FACS buffer to wash, spin down at 1500RPM for 5 mins. at 4 C, and tip off supernatant, blotting on tissue. 16. Add 50ul diluted Fc block per well, mix and incubate for 15 mins. on ice. See below for example calculation for 20 wells:- (always make extra to account for pipetting loss/error) Reageant Dilution Volume (ul) Fc block 1:100 10 FACS Buffer - 990 --> At this point, start preparing the live/dead fractions for the single cell viability dye only control stain (see Appendix C). 2

17. Add 150ul FACS buffer to wash, spin down at 1500RPM for 5 mins. at 4 C, and tip off supernatant, blotting on tissue. 18. Add 50ul/well of diluted CD8 and/or CD4/CD3 antibody together with live/dead viability stain (or viability stain alone for viability compensation well), mix and incubate for 30 mins. on ice IN THE DARK. See below for example calculation for 20 wells:- (always make extra to account for pipetting loss/error) Reageant Dilution Volume (ul) CD8-AF488 1:400 2.5 Viability Dye-eF450 0.1ul per well 2.0 FACS Buffer - 995.5 19. Wash 150ul FACS buffer, spin down at 1500RPM for 5 mins. at 4 C, and tip off supernatant, blotting on tissue. 20. Wash 200ul FACS buffer, spin down at 1500RPM for 5 mins. at 4 C, and tip off supernatant, blotting on tissue. 21. Fix with 100ul Cytofix-Cytoperm (undiluted), mix well and incubate for 20 mins. on ice IN THE DARK. 22. Wash in 100ul permwash buffer, spin down at 1500RPM for 5 mins. at 4 C, and tip off supernatant, blotting on tissue. Pellets should now be transparent. 23. Wash in 200ul permwash buffer, spin down at 1500RPM for 5 mins. at 4 C, and tip off supernant, blotting on tissue. 24. Add 50ul diluted intracellular antibody cocktail for 30 mins. on ice IN THE DARK. See below for example calculation for 20 wells:- Reageant Dilution Volume (ul) IFNg-AF647 1:200 5.0 TNFa-PE-Cy7 1:300 3.3 IL-2-PE 1:200 5.0 Permwash buffer - 986.7 25. Add 150ul permwash buffer, spin down at 1500RPM for 5 mins. at 4 C, and tip off supernatant, blotting on tissue. 26. Add 200ul permwash buffer, spin down at 1500RPM for 5 mins. at 4 C, and tip off supernatant, blotting on tissue. 27. Final resuspension in 200ul FACS buffer. Keep cells on ice and in dark until running on flow cytometer. 3

Fortessa HTS loader settings: Mode: standard Sample rate: 2 ul/sec (decrease if needed) Sample volume: 140ul Mixing volume: 80ul Mixing speed: 200 ul/sec (if too high the content of wells might spill over, and risk air bubbles) Number of mixes: 3 Wash volume: 400 4

APPENDIX A ACK Lysis Buffer 8.29g Ammonium Chloride 1g Potassium Bicarbonate 200ul 0.5M EDTA ph8.0 800ml dh2o ph to 7.4, then make up to 1L with dh2o 10x Sterile Heparin (Stock Solution) The 10x sterile heparin stock solution should be at 400U/ml. e.g. weigh 122mg heparin sodium salt (from porcine intestinal mucosa) at 164 USP units/mg. Resuspend in 50ml PBS = 400U/ml solution. Filter stock solution through a 0.45um filter. This can be stored for several months at 4 C. The 10x stock solution is diluted 10-times in PBS to generate the 40U/ml working stock that we collect blood into. You can store the working stock for several months at 4 C. DC Medium 50ml FCS 5ml P/S (Pencillin/Streptomycin) 5ml NEAA (Non-Essential Amino Acids) 5ml HEPES 1.9ul 14.3M 2-beta-mercaptoethanol 500ml RPMI (with L-Glut already added) 5

APPENDIX B Preparing Negative Control Master Mix Add 100ul/well of DMSO diluted in DC medium at the highest concentration used in your test peptide stimulations e.g. if you stimulated some wells with 1ul SIINFEKL peptide in 5 ml DC medium, and some wells with 10uL m38 peptide in 5 ml, make up your negative control by adding 10ul of DMSO to 5ml DC medium. Preparing Positive Control PMA/Ionomycin Master Mix 1. Phorbol 12-myristate 13-acetate (PMA) powder (Sigma #P-1585) a. Reconstitute in DMSO at 0.1 mg/ml. b. Store small aliquots (eg, 20 ul) at 20 C; do not refreeze aliquots after thawing. 2. Ionomycin powder (Sigma #I-0634) a. Reconstitute in EtOH at 0.5 mg/ml. b. Store at 20 C. 3. Mix PMA solution at a final concentration of 100ng/ml of cell suspension (in DC medium) with fresh ionomycin solution at a final concentration of 1ug/ml of cell suspension (in DC medium). See example calculation below for 20 wells:- Reageant Volume (ul) PMA (0.1mg/ml) 1.0 Ionomycin (0.5mg/ml) 2.0 DC medium 997.0 Preparing Test Peptide Master Mix Check stock concentration of peptides you may need to adjust the following calculations for different peptides. Default final peptide concentrations are 1ug/ml. Some common peptides are listed below:- CD8+ Peptides in H-2 b haplotype (i.e. C57BL/6): Peptide MHC Protein Peptide Sequence Stock Concentration Final Concentration H2-Kb hdct 180-188 SVYDFFVWL 5mg/ml 2ug/ml H2-Db hgp100 25-33 KVPRNQDWL 10mg/ml 1ug/ml H2-Db mgp100 25-33 EGSRNQDWL 10mg/ml 1ug/ml H2-Kb OVA 257-264 SIINFEKL 5mg/ml 1ug/ml H2-Kb βgal 97-104 DAPIYTNV 5mg/ml 1ug/ml H2-Kb VSV N 52-59 RGYVYQGL 10mg/ml 1ug/ml H2-Kb FMT N 301-309 AVVLMFAQC 10mg/ml 1ug/ml H2-Kb MCMV m38 316-323 SSPPMFRV 10ug/ml H2-Kb AdV DBP 418-426 FALSNAEDL 20ug/ml CD8+ Peptides in H-2 d Haplotype (i.e. Balb/C): Peptide MHC Protein Peptide Sequence Stock Concentration Final Concentration CT26 Tumour Cells SPSYVYHQF 1ug/ml H2-Dd VSV N 275-283 MPYLIDFGL 1ug/ml pp65 15-23 LGPISGHVL 6

APPENDIX C Preparing Compensation Controls for Each Antibody in Your Staining Cocktail 1. Vortex compensation beads (ebioscience One Comp ebeads #01-1111-42). Label a separate tube for each antibody/fluorophore used in your experiment. 2. Add 1 drop beads per tube. 3. Add 1ul of the exact antibody-fluorophore that you used to stain your cells e.g. add 1ul IFNg-AF647 for your AF647 compensation control. Vortex. Incubate at RT for 15-30 mins. IN THE DARK. 4. Wash with 2ml FACS buffer, pellet by centrifugation at 1500RPM for 5 mins. Pour off supernatant. 5. Resuspend bead pellet in 200-350ul FACS buffer. Vortex. Preparing Viability Dye Single Stain Cell Controls Plate two extra wells from your naive mouse for this control. The well receives stimulation as per the PMA/ionomycin control. During the Fc block incubation (step #16), resuspend one well (the 'dead' well) in 200ul FACS buffer, place into a 1.5ml microfuge tube and heat to 100 C (in a heat block) for 2 min. The contents of the second 'live' well stay in the plate during this heating step. Remove the tube from the heat block, and allow to cool on the bench for 5-10 min. (so you don't scorch the live cell fraction when you mix the two together). Place the 'dead' fraction back into its own well, and spin down during the post-fc block wash. Resuspend 'dead' well in 50ul viability dye-only stain (step #18; 0.1ul in 49.9ul FACS buffer) and mix with the contents of the 'live' well. You will now have a single well for your viability dye-only control. Wash and spin the plate down as normal for step #18. No further fluorescently-labelled antibodies are added to this well at any stage, but the well does receive cytofix-cytoperm, washes etc. in the same way as other samples. 7

APPENDIX D Converting Frequencies to Absolute Numbers If the volume of blood collected from each mouse is known, PBS-treated control mice were included in the experiment and samples were processed in parallel and run through the flow cytometer in the same final volume for exactly the same length of time, then frequencies can be converted to physiologically-relevant absolute numbers as follows: 1. Add up the total # of lymphocyte events collected on the flow cytometer. Calculate the mean # of lymphocyte events collected from the PBS control group. 2. Calculate the adjustment factor that converts the mean # of lymphocyte events to the expected physiological # (=7.07x10 3 /µl blood; Charles River Laboratories dataset for female C57BL/6 mice; BALB/c=6.69x10 3 /µl blood). Multiply all raw lymphocyte numbers by this adjustment factor. 3. All lymphocyte subset frequencies can then be multiplied by the adjusted lymphocyte # to determine their respective # s. To estimate total # of cells circulating in the mouse (i.e. scale up from your nonterminal blood volume to the total blood volume of the mouse), assume each mouse has a total blood volume of 1.89 ml (based on the weight chart for female C57BL/6 mice provided by Charles River Labs and Methods of blood collection in the mouse J. Hoff, Lab Animal, 29:10, pp. 47-53; assumptions: an average age of 10 weeks at the time of sampling, a mean body weight of 21 g and a mean blood volume of 9 % of body weight). 8

APPENDIX E Common Cell Phenotype Markers T Cells Myeloid Dendritic Cells CD3 + CD11b + CD4 + and/or CD8 + CD11c hi Regulatory T Cells B220 - CD3 + Gr-1 - CD4 + NK1.1 - Foxp3 + CD3 - CD25 + MHC class II + CTLA4 + Macrophages/Monocytes GITR + Gr-1 - CD127 lo CD11b lo/mid B Cells CD11c + B220 + B220 - CD19 + F40/80 + CD3 - NK1.1 - NK Cells CD3 - CD3 - MHC class II + NK1.1/DX5 + Myeloid Suppressor Cells Plasmacytoid Dendritic Cells Gr-1 + CD11b lo/mid CD11b + CD11c mid CD80 + B220 + CD40 - Gr-1 + B220 - NK1.1 - CD86 - CD3 - IKDC s MHC class II + B220 + CD19 - CD11c + NK T Cells MHC class II + CD3 + NK1.1/DX5 + NK1.1/DX5 + CD3 - Gr-1-9