MBTP 26: Immunophenotyping of regulatory T cells from human PBMCs using flow cytometry
Miltenyi Biotec-tested panel 26 (MBTP 26)
Miltenyi Biotec-tested panel 26 (MBTP 26)
This application protocol describes the simultaneous quantification of intracellular and surface markers for easy enumeration of regulatory T cells from human PBMCs using flow cytometry.
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 staining cocktail with all antibodies was prepared according to the dilutions mentioned in the respective antibody data sheets.
- 100 µL antibody staining cocktail 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.
- Fixation and Permeabilization solution was prepared by diluting Fixation/Permeabilization Solution 1 1:3 with Fixation/Permeabilization Solution 2.
- 1× solution of Permeabilization Buffer was prepared by diluting 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 per 106 cells 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 10 µL of Tandem Signal Enhancer and 88 µL of cold 1× Permeabilization Buffer.
- 2 µL of FoxP3 Antibody, anti-human, Vio® 667, REAfinity™ was added to the cells.
- 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 PEB buffer and flow cytometric analysis was performed using the MACSQuant® Analyzer.
Antibody panel for regulatory T cells
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| CD45 | REA747 | Lymphocytes | VioBlue® | 450/50 (violet) |
| CD4 | REA623 | T helper cells | VioGreen™ | 525/50 (violet) |
| CD25 | REA570 | Regulatory T cells | Vio® Bright B515 | 525/50 (blue) |
| CD127 | REA614 | Regulatory T cells | PE | 585/40 (blue) |
| FoxP3 | REA944 | Transcription factor of regulatory T cells | Vio 667 | 655-730 (blue) |
Gating strategy
REA570
FC
EUR 176,00
Materials
Antibody panel for regulatory T cells
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| CD45 | REA747 | Lymphocytes | VioBlue® | 450/50 (violet) |
| CD4 | REA623 | T helper cells | VioGreen™ | 525/50 (violet) |
| CD25 | REA570 | Regulatory T cells | Vio® Bright B515 | 525/50 (blue) |
| CD127 | REA614 | Regulatory T cells | PE | 585/40 (blue) |
| FoxP3 | REA944 | Transcription factor of regulatory T cells | Vio 667 | 655-730 (blue) |
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
- Treg Phenotyping Kit, anti-human, REAfinity™ (# 130-122-994)
- (Optional) Transcription Factor Staining Buffer Set (# 130-122-981)
- (Optional) Tandem Signal Enhancer, human (# 130-099-887)
- 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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REA570
FC
EUR 176,00
