MBTP 28: Immunophenotyping of virus-specific T cells from human blood using flow cytometry

Miltenyi Biotec-tested panel 28 (MBTP 28)

This application protocol describes the flow cytometric analysis of virus-specific T cells after enrichment of IFN-γ⁺ cells from human blood. IFN-γ⁺ cells were enriched using the CliniMACS® Cytokine Capture System (IFN-gamma) to obtain viable single cell suspensions ready for downstream applications, such as flow cytometric analysis.

Print protocol

Protocol

Staining protocol

Surface staining

Volumes given below were 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 master mix with all fluorochrome-conjugated antibodies except IFN-γ Antibody, anti-human, PE was prepared according to the dilution listed in the respective antibody data sheets.
▲Note: IFN-γ is an intracellular marker and will be detected after fixation and permeabilization of cells.
100 µL antibody master mix was added to the cells.
Cells were resuspended and incubated for 10 minutes in the dark at 4 °C.
Cells were washed by addition of 1 mL of PEB buffer and centrifuged at 300×g for 5 minutes at 4 °C. Supernatant was aspirated completely.
Intracellular staining was performed.

Intracellular staining

Reagents were prepared freshly. Failure to do so may lead to sub-optimal results.
The required total buffer volumes were calculated beforehand; volumes will depend on the number of cells to be analyzed as well as the number of tests to be performed.

Cells previously stained for surface markers were resuspended in 250 µL of Inside Fix from the Inside Stain Kit.
Cells were mixed well and incubated for 20 minutes at room temperature.
Cells were centrifuged for 5 minutes at 300×g.
Supernatant was aspirated completely and the pellet was resuspended in 1 mL of PEB buffer.
Cells were centrifuged for 5 minutes at 300×g and supernatant was aspirated completely.
IFN-γ Antibody, anti-human, PE was diluted in Inside Perm according to the dilution mentioned in the respective data sheet to a final volume of 100 µL and added to the cells.
Cells were incubated for 10 minutes at room temperature.
1 mL of Inside Perm buffer was added and cells were centrifuged for 5 minutes at 300xg. Supernatant was aspirated completely.
Cells were resuspended in 0.5–1 mL of PEB buffer and flow cytometric analysis was performed using the MACSQuant® Analyzer.

Flow cytometric analysis

Gating strategy showing the analysis of virus-specific T cells from human blood. IFN-γ enriched cells were stained using the previously described panel. Debris was excluded using FSC-H/SSC-H gating (A). Cells were analyzed for viable cells using 7-AAD-negative gating (data not shown). Viable cells were gated on CD45 and CD3 expression to identify T cells (B, C). T helper cells and cytotoxic T cells were identified based on expression of CD4 and CD8 (D). In addition, naive, central memory (CM), effector (E), and effector memory (EM) T cells were detected within CD3⁺ cells by analysis of CD45RO and CD62L expression (E). Moreover, CD45RO and CD62L expression of CD3⁺ IFN-γ⁺ cells (F, I), CD3⁺ CD4⁺ IFN-γ⁺ cells (G, J), and CD8⁺ IFN-γ⁺ cells (H, K) was determined.

Materials

Reagent and instrument requirements

Antibody panel details

Specificity	Clone	Purpose	Fluorochrome	Detection filter (nm) (laser)
7-AAD Staining Solution	-	Viability	-	655–730 (blue)
CD45	REA747	Leukocytes	VioBlue®	450/50 (violet)
CD4	REA623	T helper cells	VioGreen™	525/50 (violet)
CD3	REA613	T cells	FITC	525/50 (blue)
CD8	REA734	Cytotoxic T cells	APC-Vio® 770	750 LP (red)
IFN-γ	45-15	Activation marker	PE	585/40 (blue)
CD45RO	REA611	Memory T cells	PE-Vio 770	750 LP (blue)
CD62L	145/15	Naive and memory T cells	APC	655–730 (red)

Additional reagents used

Phosphate-buffered saline (PBS), pH 7.2
PEB buffer: PBS, pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA.
Deionized or distilled water
Inside Stain Kit (# 130-090-477)
Flow cytometer, for example, MACSQuant® Analyzer 10 (# 130-096-343) or MACSQuant X (# 130-105-100)
(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.

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

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MBTP 28: Immunophenotyping of virus-specific T cells from human blood using flow cytometry

Miltenyi Biotec-tested panel 28 (MBTP 28)

Protocol

Materials

Protocol

Staining protocol

Surface staining

Intracellular staining

Flow cytometric analysis

Materials

Reagent and instrument requirements

Antibody panel details

Additional reagents used

Trademarks and disclaimers

7-AAD Staining Solution

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

CD45 Antibody, anti-human, VioBlue
^®
, REAfinity™

CD3 Antibody, anti-human, FITC, REAfinity™

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

CD45RO Antibody, anti-human, PE-Vio
^®
770
, REAfinity™

CD62L Antibody, anti-human, APC

IFN-γ Antibody, anti-human, PE

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