TITLE: Immune profiling using 16-color panel for PD patient PBMCs
AUTHORS: Weiyi Peng

[DESCRIPTION]
This is the protocol for immune profiling using 16-color panel for PD patient PBMCs.

[STEPS]
SECTION: Master tubes summary:
1. 1. Ghost dye stain: Day 1 
2. Fc blocker: Day 1 
3. Surface marker: Day 1 
4. Intracellular markers: Day 1 
5. Foxp3 fix/perm buffer: Day 2 
6. FoxP3 wash buffer: Day 1
SECTION: Day 1 (the day before staining) for 16 colors staining:
2. Compo-Beads staining:
SECTION: Day 1 (the day before staining) for 16 colors staining:
2.1. 1. Label 1.5ml tubes with Compo-beads control IDs
SECTION: Day 1 (the day before staining) for 16 colors staining:
2.2. 2. Add 1 drop of Compo-bead into corresponding tube (1 drop for each tube, totally 15 drops)
SECTION: Day 1 (the day before staining) for 16 colors staining:
2.3. 3. Add antibody into corresponding tubes
SECTION: Day 1 (the day before staining) for 16 colors staining:
2.4. 4. Vortex well, and incubate at 4°C for 30 min, avoid light
SECTION: Day 1 (the day before staining) for 16 colors staining:
2.5. 5. Wash with 1 mL Stain Buffer
SECTION: Day 1 (the day before staining) for 16 colors staining:
2.6. 6. Spin at 1500 rpm for 5 min
SECTION: Day 1 (the day before staining) for 16 colors staining:
2.7. 7. Dump the supernatant, and directly tap on kim-wiper once gently
SECTION: Day 1 (the day before staining) for 16 colors staining:
2.8. 8. Resuspend into 500 µl Stain Buffer
SECTION: Day 1 (the day before staining) for 16 colors staining:
2.9. 9. Wrap all the tubes with plastic wrap to prevent evaporation, and cover with foil
SECTION: Day 1 (the day before staining) for 16 colors staining:
2.10. 10. Store at 4°C
SECTION: Day 1 (the day before staining) for 16 colors staining:
3. Ghost dye bead staining
SECTION: Day 1 (the day before staining) for 16 colors staining:
3.1. 1. Add 2x drops of ArC positive beads (green top) into the FACS tube, labeled as GB-16 for Ghost dye bead staining
SECTION: Day 1 (the day before staining) for 16 colors staining:
3.2. 2. Add 0.5 µl of Ghost Violet 510 into the tube
SECTION: Day 1 (the day before staining) for 16 colors staining:
3.3. 3. Vortex well, and incubate at 4°C for 30 min
SECTION: Day 1 (the day before staining) for 16 colors staining:
3.4. 4. Wash with 1 mL Stain Buffer
SECTION: Day 1 (the day before staining) for 16 colors staining:
3.5. 5. Spin at 1500 rpm for 5 min
SECTION: Day 1 (the day before staining) for 16 colors staining:
3.6. 6. Dump the supernatant, and directly tap on kim-wiper once gently
SECTION: Day 1 (the day before staining) for 16 colors staining:
3.7. 7. Resuspend into 500 µl Stain Buffer
SECTION: Day 1 (the day before staining) for 16 colors staining:
3.8. 8. Wrap all the tubes with plastic wrap to prevent evaporation, and cover with foil
SECTION: Day 1 (the day before staining) for 16 colors staining:
4. Prepare the surface antibody cocktails using brilliant buffer
SECTION: Day 1 (the day before staining) for 16 colors staining:
3.9. 9. Store at 4°C
- Note: Add 2x drops of ArC negative beads (white top) into the tube right before running FACS. 
SECTION: Day 1 (the day before staining) for 16 colors staining:
4.1. 

    Prepare full cocktails: Mix antibodies together by the volume calculated.
     Surface      Markers      Volume
  (µl)      *N      Brilliant
  buffer (µl)      Aliquot        Y      CD19
        5             10      Total XXX µl;     64ul for each sample        Y      CD3      5               Y      CD11c      5               Y      CXCR5(CD185)      5               Y      CD103      5               Y      PD-1
  (CD279)      5               Y      CD45      5               Y      CD4      5               Y      CD8      5               Y      CD11b      1               Y      CD56      5               Y      CD25      3               Total      54             10*N=   
 
 
 
 
SECTION: Day 1 (the day before staining) for 16 colors staining:
4.2. 2. Vortex well, and store at 4oC
SECTION: Day 1 (the day before staining) for 16 colors staining:
5. Prepare the intracellular staining cocktails using Foxp3 wash buffer
SECTION: Day 1 (the day before staining) for 16 colors staining:
5.1. Full Intracellular panel for 82 samples
 
     IC Antibody      Volume/ Sample (µl)      *N      Foxp3 wash buffer        FoxP3 eFluor 450      5             35*N µl        Ki67 APC      5               CD107a PE      5               Total      XXX µl; 50ul / sample   
Vortex well, and store at 4oC
SECTION: Day 1 (the day before staining) for 16 colors staining:
6. Prepare the Fc blocker: 1:100 dilution with stain buffer
SECTION: Day 1 (the day before staining) for 16 colors staining:
6.1. 
     Antibody      Volume/ Sample (µl)      *N      For each sample        CD16/CD32      1             100 µl        Stain buffer      99          

Vortex well and store at 4oC 
 
 
SECTION: Day 1 (the day before staining) for 16 colors staining:
7. Ghost dye master tube: 0.5ul Ghost dye + 100ul PBS for each sample
SECTION: Day 1 (the day before staining) for 16 colors staining:
7.1. 1. Remove Ghost Dye vial from freezer and allow to equilibrate to room temperature.
SECTION: Day 1 (the day before staining) for 16 colors staining:
7.2. 2. Quick spin Ghost Dye vial before opening.
SECTION: Day 1 (the day before staining) for 16 colors staining:
7.3. 3. Make Ghost Dye staining solution, and vortex well
- Note: Ghost Dyes are formulated in DMSO, pipet carefully and slowly.

       Volume/ Sample (µl)      *N      For each sample        Ghost
  dye Volume/ Sample (µl)      0.5             100 µl        PBS      100          

SECTION: Day 1 (the day before staining) for 16 colors staining:
8. FoxP3 Fix/Perm buffer: 1:4 dilution; 100ul for each sample; prepare it on day 2
SECTION: Day 1 (the day before staining) for 16 colors staining:
9. FoxP3 wash buffer: 1:10 dilution by water
SECTION: Day 1 (the day before staining) for 16 colors staining:
10. Prepare 15ml columns, FACS tubes with cell strainer snap cap and 78 frozen vials; label them accordingly.
SECTION: Day 2
11. Control cells:
SECTION: Day 2
11.1. 1. Add 9ml RPMI medium into each 15mL tube.
SECTION: Day 2
11.2. 2. Thaw one vial of PBMCs from -80°C at 37°C until it has a small piece of ice at a time. Add PBMCs into the 15mL tube.
SECTION: Day 2
11.3. 3. Count cell numbers, and record the volume, viability and concentration.
SECTION: Day 2
11.4. 4. Spin down PBMCs for 5min at 1500rpm, resuspend in RPMI medium to get the final concentration of 10x106/ml.
SECTION: Day 2
11.5. 5. Add 100ul PBMCs into the V-bottom plates; Will need two wells, one is for no stain control (A), and one for Ghost dye only control (B)
SECTION: Day 2
12. PBMC Samples:
SECTION: Day 2
12.1. 1. Take all the PBMCs from LN2 to -80°C.
SECTION: Day 2
12.2. 2. Add 9ml RPMI medium into each 15mL tube.
SECTION: Day 2
12.3. 3. Thaw maximum 5 vials of PBMCs from -80°C at 37°C until it has a small piece of ice at a time. Add PBMCs into the 15mL tube.
SECTION: Day 2
12.4. 4. Count cell numbers, and record the volume, viability and concentration.
SECTION: Day 2
12.5. 5. Spin down PBMCs for 5min at 1500rpm, resuspend in RPMI medium to get the final concentration of 10x106/ml.
SECTION: Day 2
12.6. 6. Add 100ul PBMCs into the V-bottom plates; freeze the rest cells back using Mr. Frosty™ Freezing Container for future use.
SECTION: Day 2
12.7. 7. Spin down PBMCs in V-bottom 96-well plate (26 samples for each group), discard the supernatant, add 200ul Stain buffer into the each well to wash the cells.
SECTION: Day 2
12.8. 8. Spin down PBMCs for 5min at 1500rpm, discard the supernatant.
SECTION: Day 2
13. Ghost dye staining: Include Ghost dye only control
SECTION: Day 2
13.1. 9. Add 100 µl of the Ghost Dye staining solution into cells (Include Ghost dye only control).
SECTION: Day 2
14. 10.  Incubate cells for 30 minutes at 4°C protected from light.
SECTION: Day 2
14.1. 11.  Wash cells with 100ul Stain buffer. 
SECTION: Day 2
14.2. 12.  Spin down at 1500 rpm for 5 min
SECTION: Day 2
14.3. 13.  Aspirate the supernatant.
SECTION: Day 2
14.4. The samples are ready for Fc blocking except Ghost dye only control. For Ghost dye only control, store cells at 4oC for future permeabilization.
SECTION: Day 2
15. FC Blocking: For PD patient PBMC samples
SECTION: Day 2
15.1. 14.  Add 100uL of FC blocker into cells 
SECTION: Day 2
15.2. 15.  Incubate cells at RT for 10 min
SECTION: Day 2
15.3. 16.  Wash the samples with 100ul Stain buffer
SECTION: Day 2
15.4. 17.  Spin down at 1500 rpm for 5 min (with Eppendorf tube centrifuge)
SECTION: Day 2
15.5. 18.  Aspirate the supernatant (the pellets are ready for surface staining)
SECTION: Day 2
16. Surface staining: For PD patient PBMC samples
SECTION: Day 2
16.1. 19.  Add 64 µl of surface staining cocktail from yesterday into samples, and mix them well
SECTION: Day 2
16.2. 20.  Incubate at 4oC for 30 min, protected from the light
SECTION: Day 2
16.3. 21.  Wash with 100ul Stain Buffer, spin down at 1500 rpm for 5 min
SECTION: Day 2
16.4. 22.  Aspirate the supernatant (the pellets are ready for the cell permeabilization)
SECTION: Day 2
17. Cell Permeabilization: Include no stain control and Ghost dye only control
SECTION: Day 2
17.1. 23.  Prepare Foxp3 perm buffer (1:4 dilution)
SECTION: Day 2
17.2. 24.  Add 100uL of Foxp3 perm buffer into each tube, and mix them well
SECTION: Day 2
17.3. 25.  Incubate cells at RT for 60 min
SECTION: Day 2
17.4. 26.  Wash with 100ul Foxp3 wash buffer
SECTION: Day 2
17.5. 27.  Spin down at 1500 rpm for 5 min 
SECTION: Day 2
17.6. 28.  Aspirate the supernatant (the pellets are ready for the intracellular staining).
SECTION: Day 2
18. Intracellular staining:
SECTION: Day 2
18.1. 29.  Add 50 µl of the intracellular antibody cocktails from yesterday, and mix them well
SECTION: Day 2
18.2. 30.  Incubate at 4 oC for 30 min
SECTION: Day 2
18.3. 31.  Wash with 100ul Foxp3 wash Buffer
SECTION: Day 2
18.4. 32.  Spin down at 1500 rpm for 5 min
SECTION: Day 2
18.5. 33.  Aspirate the supernatant
SECTION: Day 2
18.6. 34.  Resuspend the pellets into 200ul stain Buffer
SECTION: Day 2
18.7. 35.  Vortex, and store at 4oC
SECTION: Analyze the samples using The BD LSRFortessa™ Cell Analyzer.
19. Gating strategy


T cell: CD45+CD3+CD19-/CD45+
CD4 T cell: CD45+CD3+CD19-CD4+ CD8-/ T cell
Treg cell: CD45+CD3+CD19-CD4+ CD8- CD25+ Foxp3+/CD4 T
P-CD4 cell: CD45+CD3+CD19-CD4+ CD8-Ki67+ /CD4 T
CD8 T cell: CD45+CD3+CD19-CD4- CD8+/T cell
P-CD8 T cell: CD45+CD3+CD19-CD4- CD8+Ki67+/CD8 T cell
CD8-CD103 cell: CD45+CD3+CD19-CD4- CD8+CD103+/CD8 T cell
CD8-CD107 cell: CD45+CD3+CD19-CD4- CD8+CD107+ (?)/CD8 T cell
CD8-PD1 cell: CD45+CD3+CD19-CD4- CD8+PD-1+ (?)/CD8 T cell
B cell: CD45+CD3-CD19+/CD45
P-B cell: CD45+CD3-CD19+Ki67+/B cell
NK cell: CD45+CD3-CD19-CD56+CD11C-/non T and non B
P-NK cell: CD45+CD3-CD19-CD56+CD11C-Ki67+/NK
DC cell: CD45+CD3-CD19-CD56-CD11C+/non T and non B
P-DC cell: CD45+CD3-CD19-CD56-CD11C+/NK
Monocyte: CD45+CD3-CD19-CD11b+CD11C-/non T, B, DC, NK


ASAP 77 PBMCs – Columbia cohort:
1. #UPL222C is missing (Well D4, no tube received from Columbia) 
2. Wells A5, B5, C5, D5, E5, F5, G5, H5 have poor results (removed Treg, CD103, CD107, Ki67,
Monocytes)