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

MBTP 54 = Miltenyi Biotec-tested panel 54

This application protocol describes the analysis of human CD4⁺ and CD8⁺ T cell activation markers after T cell stimulation. To demonstrate this state-of-the-art panel for CD4 T cell activation as well as CD8 T cell activation, PBMCs were stimulated for 48 hours using Miltenyi Biotec’s T Cell TransAct™, human. The activation state was defined by the expression level of HLA-DR, CD25, CD69, and CD38.

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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 CD4⁺ and CD8⁺ T cell activation

Specificity	Clone	Purpose	Fluorochrome	Detection filter (nm) (laser)
7-AAD*	-	Viability	-	655-730 (blue)
HLA-DR	REA805	T cell activation	FITC	525/50 (blue)
CD4*	REA623	Helper T cells	VioGreen™	525/50 (violet)
CD8*	REA734	Cytotoxic T cells	APC-Vio® 770	750 LP (red)
CD38	REA572	T cell activation	APC	655-730 (red)
CD3	REA613	T cells	VioBlue®	450/50 (violet)
CD25	REA570	T cell activation	PE	585/40 (blue)
CD69	REA824	T cell activation	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 48 hours after stimulation.

Among CD4⁺ T cells, the state of activation was defined by HLA-DR (A), CD25 (B), CD69 (C), and CD38 expression (D).

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Figure 3: Gating strategy for the analysis of human CD8⁺ T cell activation 48 hours after stimulation.

Among CD8⁺ T cells, the state of activation was defined by HLA-DR (A), CD25 (B), CD69 (C), and CD38 expression (D).

View details

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

130-112-615

CD69 Antibody, anti-human, PE-Vio
^®
770
, REAfinity™

REA824

100

tests

in 200 µL (1)

USD 345.00

130-111-788

HLA-DR Antibody, anti-human, FITC, REAfinity™

REA805

FC, MICS, IF, IHC

100

tests

in 200 µL (1)

USD 249.00

130-113-991

CD38 Antibody, anti-human, APC, REAfinity™

REA572

FC, MICS, IF, IHC

tests

in 60 µL (1)

USD 132.00

130-113-230

CD4 Antibody, anti-human, VioGreen™, REAfinity™

REA623

100

tests

in 200 µL (1)

USD 361.00

130-113-286

CD25 Antibody, anti-human, PE, REAfinity™

REA570

100

tests

in 200 µL (1)

USD 297.00

130-114-519

CD3 Antibody, anti-human, VioBlue
^®
, REAfinity™

REA613

100

tests

in 200 µL (1)

USD 317.00

Materials

Reagent and instrument requirements

Antibody panel for CD4⁺ and CD8⁺ T cell activation

Specificity	Clone	Purpose	Fluorochrome	Detection filter (nm) (laser)
7-AAD*	-	Viability	-	655-730 (blue)
HLA-DR	REA805	T cell activation	FITC	525/50 (blue)
CD4*	REA623	Helper T cells	VioGreen™	525/50 (violet)
CD8*	REA734	Cytotoxic T cells	APC-Vio® 770	750 LP (red)
CD38	REA572	T cell activation	APC	655-730 (red)
CD3	REA613	T cells	VioBlue®	450/50 (violet)
CD25	REA570	T cell activation	PE	585/40 (blue)
CD69	REA824	T cell activation	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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Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use.

If not otherwise specified, all marks displayed on the Miltenyi Biotec Internet site and documents are subject to the trademark rights of Miltenyi Biotec, including each of Miltenyi Biotec's primary brands, its model name plates, and its corporate logos and emblems.

For copyright note and a complete list of trademarks visit www.miltenyibiotec.com/legalnotes.

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

130-112-615

CD69 Antibody, anti-human, PE-Vio
^®
770
, REAfinity™

REA824

100

tests

in 200 µL (1)

USD 345.00

130-111-788

HLA-DR Antibody, anti-human, FITC, REAfinity™

REA805

FC, MICS, IF, IHC

100

tests

in 200 µL (1)

USD 249.00

130-113-991

CD38 Antibody, anti-human, APC, REAfinity™

REA572

FC, MICS, IF, IHC

tests

in 60 µL (1)

USD 132.00

130-113-230

CD4 Antibody, anti-human, VioGreen™, REAfinity™

REA623

100

tests

in 200 µL (1)

USD 361.00

130-113-286

CD25 Antibody, anti-human, PE, REAfinity™

REA570

100

tests

in 200 µL (1)

USD 297.00

130-114-519

CD3 Antibody, anti-human, VioBlue
^®
, REAfinity™

REA613

100

tests

in 200 µL (1)

USD 317.00

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

MBTP 54 = Miltenyi Biotec-tested panel 54

Protocol

Materials