Aug 16, 2023
Immune profiling using 16-color panel for PD patient PBMCs
Protocol Citation: Daniel Choo: Immune profiling using 16-color panel for PD patient PBMCs. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7pp13gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 16, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86579
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network
Grant ID: ASAP-000312
Abstract
This is the protocol for immune profiling using 16-color panel for PD patient PBMCs.
Materials
Materials:
1. 1x PBS
2. Flow cytometry staining buffer (Stain Buffer) – 1x PBS + 2% FBS, filtered with 0.2um filter
3. Brilliant buffer plus
4. Staining Kit: eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set (00-5523-00)
5. PBMC isolated from buffy coat for no stain control and Ghost dye only control
6. 15ml tubes, frozen vials, V-bottom 96-well plates, FACS tubes with cell strainer snap cap
Samples: PD patient PBMCs; 1M cells per sample
Master tubes summary:
Master tubes summary:
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
Day 1 (the day before staining) for 16 colors staining:
Day 1 (the day before staining) for 16 colors staining:
Compo-Beads staining:
1. Label 1.5ml tubes with Compo-beads control IDs
2. Add 1 drop of Compo-bead into corresponding tube (1 drop for each tube, totally 15 drops)
3. Add antibody into corresponding tubes
4. Vortex well, and incubate at 4°C for 30 min, avoid light
5. Wash with 1 mL Stain Buffer
6. Spin at 1500 rpm for 5 min
7. Dump the supernatant, and directly tap on kim-wiper once gently
8. Resuspend into 500 µl Stain Buffer
9. Wrap all the tubes with plastic wrap to prevent evaporation, and cover with foil
10. Store at 4°C
Ghost dye bead staining
1. Add 2x drops of ArC positive beads (green top) into the FACS tube, labeled as GB-16 for Ghost dye bead staining
2. Add 0.5 µl of Ghost Violet 510 into the tube
3. Vortex well, and incubate at 4°C for 30 min
4. Wash with 1 mL Stain Buffer
5. Spin at 1500 rpm for 5 min
6. Dump the supernatant, and directly tap on kim-wiper once gently
7. Resuspend into 500 µl Stain Buffer
8. Wrap all the tubes with plastic wrap to prevent evaporation, and cover with foil
9. Store at 4°C
- Note: Add 2x drops of ArC negative beads (white top) into the tube right before running FACS.
Prepare the surface antibody cocktails using brilliant buffer
- Prepare full cocktails: Mix antibodies together by the volume calculated.
| A | B | C | D | E | F | |
| 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= |
2. Vortex well, and store at 4oC
Prepare the intracellular staining cocktails using Foxp3 wash buffer
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
Prepare the Fc blocker: 1:100 dilution with stain buffer
| Antibody | Volume/ Sample (µl) | *N | For each sample | |
| CD16/CD32 | 1 | 100 µl | ||
| Stain buffer | 99 |
Vortex well and store at 4oC
Ghost dye master tube: 0.5ul Ghost dye + 100ul PBS for each sample
1. Remove Ghost Dye vial from freezer and allow to equilibrate to room temperature.
2. Quick spin Ghost Dye vial before opening.
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 |
FoxP3 Fix/Perm buffer: 1:4 dilution; 100ul for each sample; prepare it on day 2
FoxP3 wash buffer: 1:10 dilution by water
Prepare 15ml columns, FACS tubes with cell strainer snap cap and 78 frozen vials; label them accordingly.
Day 2
Day 2
Control cells:
1. Add 9ml RPMI medium into each 15mL tube.
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.
3. Count cell numbers, and record the volume, viability and concentration.
4. Spin down PBMCs for 5min at 1500rpm, resuspend in RPMI medium to get the final concentration of 10x106/ml.
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)
PBMC Samples:
1. Take all the PBMCs from LN2 to -80°C.
2. Add 9ml RPMI medium into each 15mL tube.
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.
4. Count cell numbers, and record the volume, viability and concentration.
5. Spin down PBMCs for 5min at 1500rpm, resuspend in RPMI medium to get the final concentration of 10x106/ml.
6. Add 100ul PBMCs into the V-bottom plates; freeze the rest cells back using Mr. Frosty™ Freezing Container for future use.
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.
8. Spin down PBMCs for 5min at 1500rpm, discard the supernatant.
Ghost dye staining: Include Ghost dye only control
9. Add 100 µl of the Ghost Dye staining solution into cells (Include Ghost dye only control).
10. Incubate cells for 30 minutes at 4°C protected from light.
11. Wash cells with 100ul Stain buffer.
12. Spin down at 1500 rpm for 5 min
13. Aspirate the supernatant.
The samples are ready for Fc blocking except Ghost dye only control. For Ghost dye only control, store cells at 4oC for future permeabilization.
FC Blocking: For PD patient PBMC samples
14. Add 100uL of FC blocker into cells
15. Incubate cells at RT for 10 min
16. Wash the samples with 100ul Stain buffer
17. Spin down at 1500 rpm for 5 min (with Eppendorf tube centrifuge)
18. Aspirate the supernatant (the pellets are ready for surface staining)
Surface staining: For PD patient PBMC samples
19. Add 64 µl of surface staining cocktail from yesterday into samples, and mix them well
20. Incubate at 4oC for 30 min, protected from the light
21. Wash with 100ul Stain Buffer, spin down at 1500 rpm for 5 min
22. Aspirate the supernatant (the pellets are ready for the cell permeabilization)
Cell Permeabilization: Include no stain control and Ghost dye only control
23. Prepare Foxp3 perm buffer (1:4 dilution)
24. Add 100uL of Foxp3 perm buffer into each tube, and mix them well
25. Incubate cells at RT for 60 min
26. Wash with 100ul Foxp3 wash buffer
27. Spin down at 1500 rpm for 5 min
28. Aspirate the supernatant (the pellets are ready for the intracellular staining).
Intracellular staining:
29. Add 50 µl of the intracellular antibody cocktails from yesterday, and mix them well
30. Incubate at 4 oC for 30 min
31. Wash with 100ul Foxp3 wash Buffer
32. Spin down at 1500 rpm for 5 min
33. Aspirate the supernatant
34. Resuspend the pellets into 200ul stain Buffer
35. Vortex, and store at 4oC
Analyze the samples using The BD LSRFortessa™ Cell Analyzer.
Analyze the samples using The BD LSRFortessa™ Cell Analyzer.
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)