TITLE: Intracellular Cytokine Staining
AUTHORS: Kristin Anderson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines the steps used for Intracellular cytokine staining.</div><div class = "text-block"><span>In brief: Cells were treated with protein transport inhibitor containing Brefeldin A (GolgiPlug; cat: 555029, BD) and plated at 1e6 cells per well in a 96-well flat bottom plate. Cells were stimulated for 5 hours at 37°C with either T cell media, Phorbol 12-Myristate 13-Acetate (Sigma, cat: P8139) (used at 20ng/mL) and Ionomycin (Sigma, cat: I0634)(used at 1mg/mL), or 1mg/mL of peptide (as indicated). Msln</span><span style = "vertical-align:sub;">406-414</span><span> (GQKMNAQAI), OTI (SIINFEKL), LCMV</span><span style = "vertical-align:sub;">gp33-41</span><span> (KAVYNFATM), MSLN</span><span style = "vertical-align:sub;">20-28</span><span> (SLLFLLFSL), and MSLN</span><span style = "vertical-align:sub;">530-538 </span><span>(VLPLTVAEV) peptides were ordered from ELIM peptide (>80% purity). The BD Fix/Perm kit (cat: 554714) was used for intracellular staining. Cells were fixed in 0.5% paraformaldehyde until data acquisition.</span></div></div>

[STEPS]
?. Prepare a flat bottom 96 well plate. Resuspend cells at 10e6 cells/ml in T cell media. Transfer at least 350ul of cells per condition into a fresh tube (you want at least 3 replicate wells at 100ul/well for each condition).
?. Dilute Golgi Plug to 1:50 (2mg/ml) in cells for the experiment. (Once the treatment condition is added the final concentration will be 1:100 = 1mg/ml)
?. Plate 100ul of cells per well.
?. Dilute the PMA (prepare at 40ng/mL PMA so that the final concentration in the assay will be 20ng/mL) and Ionomycin (prepare at 2mg/mL Ionomycin so that the final concentration in the assay will be 1mg/mL) in one tube of media.Resuspend peptide at 2ug/100ul.
?. Add 100ul of media or PMA/I or peptide to each well.  Incubate 4-6 hours in 37°C incubator.
?. Transfer cells to U-bottom 96 well plate.         - at this step, you can combine the replicate wells if you were low on cells.
?. Centrifuge (Cfg) plate at 4oC for 2 min @809xg (with high brake) + dump supernatant.
?. Add 50ul Live/Dead stain per well (dilute 5ul of dye in 2.5ml 1x DPBS).
?. Incubate for 20-30 minutes in fridge at 4oC (dark).
?. Add 100 ul of FACS Buffer per well to wash. Cfg plate at 4oC for 2 min @809xg (with high brake) + dump supernatant.
?. Prepare cocktail of antibodies for cell surface stain. Add 50-75uL of surface stain mix per well. Resuspend pellet by pipetting up + down at least 5x.
?. Incubate plates 30 min in fridge at 4oC (dark).
?. Add 100 ul of FACS Buffer per well to wash. Cfg plate at 4oC for 2 min @809xg (with high brake) + dump supernatant.
?. Add 150 ul of FACS Buffer per well to wash a second time. Cfg plate at 4oC for 2 min @809xg (with high brake) + dump supernatant.
?. Add 150 ul of FACS Buffer per well to wash a third time. Cfg plate at 4oC for 2 min @809xg (with high brake) + dump supernatant.
?. Add 100 ul of Fixation/Permeabilization solution per well. Resuspend pellet by pipetting up + down at least 5x. Incubate plate 20 minutes at 4oC in fridge (dark).
?. Add 100 ul of Perm/Wash Buffer (PWB) per well to wash. CHANGE BREAK to SETTING 2 for all remaining centrifugation steps.
?. Cfg plate at 4oC for 2 min @809xg (with brake = 2) + dump supernatant.
?. Add 150 ul of Perm/Wash Buffer per well to wash a second time. Cfg plate at 4oC for 2 min @809xg (with brake = 2) + dump supernatant.
?. Add 150 ul of Perm/Wash Buffer per well to wash a third time. Cfg plate at 4oC for 2 min @809xg (with brake = 2) + dump supernatant.
?. Prepare cocktail of antibodies in PWB buffer for Intracellular stain. Add 50-75uL of intracellular stain mix per well. Resuspend pellet by pipetting up + down at least 5x.
?. Incubate plate 30 minutes at 4oC in fridge (dark).
?. Add 100 ul of Perm/Wash Buffer (PWB) per well to wash. Cfg plate at 4oC for 2 min @809xg (with brake = 2) + dump supernatant.
?. Add 150 ul of Perm/Wash Buffer per well to wash a second time. Cfg plate at 4oC for 2 min @809xg (with brake = 2) + dump supernatant.
?. Add 150 ul of Perm/Wash Buffer per well to wash a third time. Cfg plate at 4oC for 2 min @809xg (with brake = 2) + dump supernatant.
?. Resuspend in 200 ul of 0.5% PFA (can leave overnight at this step). Store in fridge at 4oC until acquisition. Run samples on the flow cytometer within 24 hours for best results.