Antibodies

Flow cytometry protocol – T cell exhaustion panel DOI: 10.1126/sciimmunol.aba7918

1. Resuspended PBMCs at 1 x 10⁷ cells/ml and distribute in a V-bottom 96-well plate at 100ul per well. 
2. Wash cells by adding 150 ul of fluorescence-activated cell sorting (FACS) buffer [phosphate-buffered saline (PBS) supplemented with 2% fetal bovine serum and 2 mM EDTA] and spin down for 3 minutes.  Discard supernatant without disrupting cell pellet.  Approximately 10ul volume should be left in each well.
3. Add 1ul of SA-BV421 or SA-PE conjugated MHC class I tetramer (25ug/ml) directly to the cell pellet.  Gently pipette to ensure cell pellet is resuspended.  Incubate for 20minutes at room temperature in the dark.  Skip to step 4 if not relevant. 
4. Add 150ul PBS to each well and spin at 300rpm for 3mins.  Discard supernatant.
5. Prepare fixable live dead aqua viability dye (ThermoFisher) mix in PBS - 1.5ul dye in 1ml PBS. Resuspend cell pellet in 10ul of fixable live dead aqua viablity dye mix. Gently pipette to ensure cell pellet is resuspended.  Incubate for 10minutes at room temperature in the dark.
6. Prepare chemokine staining mix in FACS buffer - 1ul CCR7 APC-Cy7 antibody (clone G043H7, BioLegend) per 10ul FACS buffer. Add 10ul of chemokine staining mix directly to the cell pellet in step 5 (do not wash after step 5).  Pipette gently to ensure cell pellet has disaggregated.  Incubate for 10 min at 37°C.
7. Prepare surface antibody staining cocktail in FACS buffer with reference to table 1.  Add 30 ul of the surface antibody cocktail to each well.  *See table 1 – surface staining for titrations*.  Be aware that final staining volume will be 50ul.
8. Incubate 20 min at room temperature in the dark.
9. Add 150ul FACS buffer and spin down at 300rpm for 3 minutes. Discard supernatant without disrupting cell pellet.
10. Add 100ul 1XFixation/Permeablization reagent (FoxP3/ Transcription Factor Staining Buffer Set - eBioscience) to the cell pellet and resuspend well. Incubate for 30 min at room temperature in the dark.
11. Wash twice by adding 150ul 1X Perm/Wash Buffer and spin down at 400rpm for 3 minutes.  Discard supernatant without disrupting cell pellet.
12. Add 50 ul of the Intracellular antibody cocktail resuspended in 1X Perm/Wash buffer.  
    *See table 1 – Intracellular staining for titrations*.
13. Incubate for 1 hour at room temperature in the dark.
14. Wash cells with 1X Perm/Wash buffer at 400rpm for 3mins.

15. Resuspend cells in 150ul PBS.  Immediately proceed to FACS analysis or store at 4°C and analyse within 24 hours.

     

Table 1. Flow cytometry antibody