MBTP 33: Immunophenotyping of activated T cells within antigen-stimulated human PBMCs using flow cytometry

Miltenyi Biotec-tested panel 33 (MBTP 33)

This application protocol describes the characterization of activated T cells within antigen-stimulated peripheral blood mononuclear cells (PBMCs) via simultaneous measurement of surface markers and intracellular cytokines using flow cytometry. Prior to antibody staining PBMCs were stimulated with a cocktail of PepTivator CMV pp65, PepTivator EBV consensus, and PepTivator AdV5 Hexon from Miltenyi Biotec.

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Protocol

Staining protocol

Labeling of cells with Viobility™ Fixable Dye

Viobility Fixable Dye was reconstituted in advance. The vial was warmed up to room temperature to avoid water condensation. One vial of lyophilized Viobility Fixable Dye was reconstituted by addition of 100 µL anhydrous DMSO to the dye vial and mixed until completely dissolved. The solution was aliquoted and stored at –20 °C under anhydrous (desiccant) conditions and protected from light for up to one month.
Volumes given below were for up to 10⁷ nucleated cells. When working with fewer than 10⁷ 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 2×10⁷ nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).

Cell number was determined.
Cells were washed by addition of 1 mL of 1× PBS and centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
Cells were resuspended in 100 µL of 1× PBS.
1 µL of Viobility Fixable Dye was added.
Samples were mixed well and incubated for 15 minutes in the dark at room temperature.
▲Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times.
Cells were washed by addition 1 mL PEB buffer and centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
Step 6 was repeated.
Surface staining was performed.

Intracellular staining

Reagents were prepared freshly. Failure to do so may lead to sub-optimal results.

2.4×10⁷ cells previously stained with Viobility™ Fixable Dye were resuspended in 3 mL PEB buffer.
3 mL Inside Fix was added.
Cells were mixed well and incubated for 20 minutes in the dark at room temperature.
Cells were centrifuged at 300×g for 10 minutes at room temperature. Supernatant was aspirated completely.
Cells were washed by addition of 5 mL PEB buffer and centrifuged at 300×g for 10 minutes at room temperature. Supernatant was aspirated completely.
Cells were resuspended in 6 mL Inside Perm and centrifuged at 300×g for 10 minutes at room temperature.
Cells were resuspended in 280 µL Inside Perm.
20 µL of cell suspension per well were transferred into a 96-well plate.
A master mix with all antibodies and Tandem Signal Enhancer, human was prepared using Inside Perm. The final dilution was 1:50 for the antibodies and 1:11 for Tandem Signal Enhancer, human. For example, for 30 µL master mix 1 µL of each antibody and 4.5 µL of Tandem Signal Enhancer, human were added to 17.5 µL of Inside Perm.
30 µL master mix was added to the cells for a final volume of 50 µL per well.
Cells were resuspended and incubated for 10 minutes in the dark at room temperature.
Cells were washed by addition of 100 µL Inside Perm and centrifuged at 300×g for 5 minutes. Supernatant was aspirated completely.
Cells were washed by addition of 200 µL Inside Perm and centrifuged at 300×g for 5 minutes. Supernatant was aspirated completely.
Cells were resuspended in 200 µL PEB buffer.
Flow cytometric analysis was performed using the MACSQuant® Analyzer 16.

Flow cytometric analysis

Figure 1: Gating strategy showing the analysis of T cell subsets within antigen-stimulated human PBMCs. Samples were initially gated on lymphocytes based on SSC-A/FSC-A gating (A). Single cells were gated using FSC-H/FSC-A and SSC-A/SSC-H gating (B, C). Dead cells and other unwanted cells were excluded (D). T cells were gated as CD3⁺ cells (E). Subsets of T cells were further gated based on CD4 and CD8 expression (F).

Figure 2: Gating strategy showing the analysis of the activation phenotype of different T cell subsets. Samples were initially gated as shown in Figure 1. The activation phenotype of the different T cell subsets CD3⁺ cells (A, B), CD4⁺ cells (C, D), CD8⁺ cells (E, F), and CD4^– CD8^– cells (G, H) was characterized based on the level of expression of the intracellular cytokines TNF-α and IFN-γ and of the surface marker CD154.

Materials

Reagent and instrument requirements

Antibody panel details

Specificity	Clone	Purpose	Fluorochrome	Detection filter (nm) (laser)
Viobility™ 405/452 Fixable Dye	-	Viability	-	450/50 (violet)
IFN-γ	REA600	T cell activation	Vio® R720	725/40 (red)
CD3	REA613	T cells	APC-Vio 770	785/62 (red)
CD4	REA623	T helper cells	Vio Bright B515	525/50 (blue)
CD8	REA734	Cytotoxic T cells	VioGreen™	525/50 (violet)
TNF-α	REA656	T cell activation	PE	579/34 (blue)
CD154	REA238	T cell activation	APC	667/30 (red)
CD14	REA599	Cell type exclusion	VioBlue®	450/50 (violet)
CD20	REA780	Cell type exclusion	VioBlue	450/50 (violet)

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)
Tandem Signal Enhancer, human (#130-099-887)
96-well plates
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)

(Optional) Reagents for antigen stimulation

PepTivator CMV pp65, human, final concentration 1:50 (# 130-093-438)
PepTivator EBV consensus, human, final concentration 1:50 (# 130-099-764)
PepTivator AdV5 Hexon, human, final concentration 1:50 (# 130-093-495)
Brefeldin A, final concentration 2 µg/ml

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MBTP 33: Immunophenotyping of activated T cells within antigen-stimulated human PBMCs using flow cytometry

Miltenyi Biotec-tested panel 33 (MBTP 33)

Protocol

Materials