MBTP 31: Immunophenotyping of human T cells via transcription factor profiling using flow cytometry
Miltenyi Biotec-tested panel 31 (MBTP 31)
Miltenyi Biotec-tested panel 31 (MBTP 31)
This application protocol describes the flow cytometric analysis of the expression of transcription factors by T cell subsets from human PBMCs.
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.
- An antibody master mix was prepared according to the dilutions mentioned in the respective antibody data sheets.
▲Note: Helios, Ki-67 and T-bet are intranuclear markers 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
- 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).
- Reagents were prepared freshly. Failure to do so may lead to sub-optimal results.
- The required total buffer volumes were calculated beforehand. Volumes depend on the number of cells to be analyzed as well as the number of tests to be performed. For more information, please refer to the data sheet of the Transcription Factor Staining Buffer Set.
- Fixation and Permeabilization solution was prepared by diluting 1:3 the Fixation/Permeabilization Solution 1 with the Fixation/Permeabilization Solution 2.
- 1× solution of Permeabilization Buffer was prepared by diluting the 10× Permeabilization buffer with deionized or distilled water.
▲Note: Before preparing the dilution make sure the buffer does not contain any precipitates. - Cells previously stained for surface markers were resuspended in 1 mL of cold, freshly prepared Fixation and Permeabilization Solution.
- Cells were mixed well and incubated for 30 minutes in the dark at 4 °C.
- Cells were washed by adding 1 mL of cold PEB buffer and centrifuged at 300×g for 5 minutes at 4 °C. Supernatant was aspirated completely.
- Cells were washed by adding 1 mL of cold 1× Permeabilization Buffer and centrifuged at 300×g for 5 minutes at 4 °C. Supernatant was aspirated completely.
- Cells were resuspended in 1× Permeabilization Buffer and the corresponding antibody volume according to the dilution mentioned in the respective data sheet was added.
- Cells were mixed well and incubated for 30 minutes in the dark at 4 °C.
- Cells were washed by adding 1 mL of cold 1× Permeabilization Buffer and centrifuged at 300×g for 5 minutes at 4 °C. Supernatant was aspirated completely.
- Cells were resuspended in 100 µL of PEB buffer and flow cytometric analysis was performed using the MACSQuant® Analyzer.
Materials
Antibody panel details
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| CD45RA | REA562 | Leukocytes | PE-Vio® 615 | 655-730 (blue) |
| CD4 | REA623 | T helper cells | APC-Vio 770 | 750 LP (red) |
| CD8 | REA734 | Cytotoxic T cells | VioGreen™ | 525/50 (violet) |
| CD25 | REA570 | Regulatory T cells | PE | 585/40 (blue) |
| CD127 | REA614 | Cell type exclusion | PE-Vio 770 | 750 LP (blue) |
| Helios | REA829 | Transcription factor | VioBlue® | 450/50 (violet) |
| Ki-67 | REA183 | Transcription factor | Vio B515 | 525/50 (blue) |
| T-bet | REA102 | Transcription factor | 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
- Transcription Factor Staining Buffer Set (# 130-122-981)
- 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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REA614
FC
CHF 358.00
REA623
FC
CHF 358.00
REA102
ICFC
CHF 330.00
REA183
ICFC
CHF 344.00
REA562
FC
CHF 358.00
