Immunophenotyping of human CD4⁺ and CD8⁺ T cell activation and exhaustion using flow cytometry

MBTP 55 = Miltenyi Biotec-tested panel 55

This application protocol describes the analysis of human CD4⁺ and CD8⁺ T cell activation and exhaustion markers after T cell stimulation. To demonstrate this state-of-the-art panel for T cell activation as well as T cell exhaustion of both CD4 T cells and CD8 T cells, PBMCs were stimulated using Miltenyi Biotec’s T Cell TransAct™, human for 72 hours. The state of activation and exhaustion was defined by CD25, CD223, CD279 (PD-1), and CD69 expression.

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Protocol

Staining 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.

Tested antibody panel

Antibody panel for T cell activation and exhaustion

Specificity	Clone	Purpose	Fluorochrome	Detection filter (nm) (laser)
7-AAD*	-	Viability	-	655−730 (blue)
CD279 (PD-1)	REA1165	T cell activation and exhaustion	Vio® Bright B515	525/50 (blue)
CD4*	REA623	Helper T cells	VioGreen™	525/50 (violet)
CD8*	REA734	Cytotoxic T cells	APC-Vio 770	750 LP (red)
CD223	REA351	T cell activation and exhaustion	APC	655−730 (red)
CD3	REA613	T cells	VioBlue®	450/50 (violet)
CD25	REA570	T cell activation and exhaustion	PE	585/40 (blue)
CD69	REA824	T cell activation and exhaustion	PE-Vio 770	750 LP (blue)

* Alternative CE-IVD/ASR reagents available.

Gating strategy

Figure 1: Gating strategy showing the identification of viable, single CD4⁺ and CD8⁺ T cells in human PBMCs. Cells were gated in an FSC-H and FSC-A dot plot to eliminate doublets (A). Lymphocytes were selected by a SSC^low gate which also excluded debris (B). Dead cells were excluded by gating on 7-AAD⁻ cells (C). CD4⁺ T cells were identified by expression of CD3 and CD4 (D). CD8⁺ T cells were identified by expression of CD3 and CD8 (E).

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Figure 2: Gating strategy for the analysis of human CD4⁺ T cell activation and exhaustion 72 hours after stimulation. Among CD4⁺ T cells, the state of activation and exhaustion was defined by CD25 (A), CD223 (B), CD279 (PD-1) (C), and CD69 expression (D).

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Figure 3: Gating strategy for the analysis of human CD8⁺ T cell activation and exhaustion 72 hours after stimulation. Among CD8⁺ T cells, the state of activation and exhaustion was defined by CD25 (A), CD223 (B), CD279 (PD-1) (C), and CD69 expression (D).

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Matching products:

130-111-568

7-AAD Staining Solution

1 mL

USD 79.00

130-110-681

CD8 Antibody, anti-human, APC-Vio
^®
770
, REAfinity™

REA734

100

tests

in 200 µL (1)

USD 340.00

130-112-804

Reagent and instrument requirements

Antibody panel for T cell activation and exhaustion

Specificity	Clone	Purpose	Fluorochrome	Detection filter (nm) (laser)
7-AAD*	-	Viability	-	655−730 (blue)
CD279 (PD-1)	REA1165	T cell activation and exhaustion	Vio® Bright B515	525/50 (blue)
CD4*	REA623	Helper T cells	VioGreen™	525/50 (violet)
CD8*	REA734	Cytotoxic T cells	APC-Vio 770	750 LP (red)
CD223	REA351	T cell activation and exhaustion	APC	655−730 (red)
CD3	REA613	T cells	VioBlue®	450/50 (violet)
CD25	REA570	T cell activation and exhaustion	PE	585/40 (blue)
CD69	REA824	T cell activation and exhaustion	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) or 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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Matching products:

130-111-568

7-AAD Staining Solution

1 mL

USD 79.00

130-110-681

CD8 Antibody, anti-human, APC-Vio
^®
770
, REAfinity™

REA734

100

tests

in 200 µL (1)

USD 340.00

130-112-804

MBTP 55 = Miltenyi Biotec-tested panel 55

Protocol

Materials