Immunophenotyping of human CD4+ and CD8+ T cell activation and differentiation using flow cytometry (panel B)
MBTP 58 = Miltenyi Biotec-tested panel 58
MBTP 58 = Miltenyi Biotec-tested panel 58
This application protocol describes the analysis of human CD4+ and CD8+ T cell activation and differentiation markers after T cell stimulation. To demonstrate this state-of-the-art panel for T cell activation as well as T cell differentiation of both CD4 T cells and CD8 T cells, PBMCs were stimulated for 48 hours using Miltenyi Biotec’s T Cell TransAct™, human. The state of activation and differentiation was defined by CD45RA, CD38, CD197 (CCR7), and HLA-DR expression.
Protocol
Surface staining
- Volumes given below are for up to 106 nucleated cells. When working with fewer than 106 cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2×106 nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).
- Cell number was determined.
- Cells were centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
- A staining cocktail with all antibodies was prepared according to the dilutions mentioned in the respective data sheets.
- 100 µL staining cocktail was added to the cells.
- Cells were resuspended and incubated for 10 minutes in the dark at room temperature .
- Cells were washed by addition of 1 mL of PEB buffer and centrifuged at 300×g for 5 minutes. Supernatant was aspirated completely.
- Cells were resuspended in 100 µL of PEB buffer. 1 µL of 7-AAD Staining Solution was added and incubated for 5 minutes in the dark at room temperature right before flow cytometric analysis using the MACSQuant® Analyzer 10.
Antibody panel for T cell activation and differentiation
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| 7-AAD* | - | Viability | - | 655−730 (blue) |
| CD45RA | REA562 | T cell activation and differentiation | FITC | 525/50 (blue) |
| CD4* | REA623 | Helper T cells | VioGreen™ | 525/50 (violet) |
| CD8* | REA734 | Cytotoxic T cells | APC-Vio® 770 | 750 LP (red) |
| HLA-DR | REA805 | T cell activation and differentiation | APC | 655−730 (red) |
| CD3 | REA613 | T cells | VioBlue® | 450/50 (violet) |
| CD38 | REA572 | T cell activation and differentiation | PE | 585/40 (blue) |
| CD197 (CCR7) | REA108 | T cell activation and differentiation | PE-Vio 770 | 750 LP (blue) |
* Alternative CE-IVD/ASR reagents available.
Gating strategy
View details
Materials
Antibody panel for T cell activation and differentiation
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| 7-AAD* | - | Viability | - | 655−730 (blue) |
| CD45RA | REA562 | T cell activation and differentiation | FITC | 525/50 (blue) |
| CD4* | REA623 | Helper T cells | VioGreen™ | 525/50 (violet) |
| CD8* | REA734 | Cytotoxic T cells | APC-Vio® 770 | 750 LP (red) |
| HLA-DR | REA805 | T cell activation and differentiation | APC | 655−730 (red) |
| CD3 | REA613 | T cells | VioBlue® | 450/50 (violet) |
| CD38 | REA572 | T cell activation and differentiation | PE | 585/40 (blue) |
| CD197 (CCR7) | REA108 | T cell activation and differentiation | PE-Vio 770 | 750 LP (blue) |
* Alternative CE-IVD/ASR reagents available.
Additional reagents used
- PEB buffer: PBS, pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA
- Flow cytometer, for example, MACSQuant® Analyzer 10 (# 130-096-343), MACSQuant X (# 130-105-100) or MACSQuant Analyzer 16 (# 130-109-803)
- (Optional) T Cell TransAct, human (# 130-111-160)
- (Optional) MACSQuant Calibration Beads (# 130-093-607)
- (Optional) MACS® Comp Bead Kit, anti-REA (# 130-104-693)
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