MBTP 33: Immunophenotyping of activated T cells within antigen-stimulated human PBMCs using flow cytometry
Miltenyi Biotec-tested panel 33 (MBTP 33)
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.
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.
Materials
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
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.
REA600
ICFC
CHF 141.00
REA613
FC
CHF 358.00
REA623
FC
CHF 448.00
