Immunophenotyping of B cells from human buffy coat using flow cytometry
MBTP 27= Miltenyi Biotec-tested panel 27
MBTP 27= Miltenyi Biotec-tested panel 27
This application protocol describes the flow cytometric analysis of B cell subsets after isolation of B cells from human buffy coat. B cells were isolated from human buffy coat using the Pan B Cell Isolation Kit, human to quickly obtain viable single cell suspensions ready for downstream applications, such as flow cytometric analysis.
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 antibodies was prepared according to the dilutions mentioned in the respective antibody data sheets.
- 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.
- Cells were resuspended in 100 µL buffer and 1 µL of 7-AAD Staining Solution was added right before flow cytometric analysis using the MACSQuant® Analyzer.
Antibody panel
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| 7-AAD Staining Solution | - | Viability | - | 655-730 (blue) |
| CD20 | REA780 | B cells | APC-Vio® 770 | 750LP (red) |
| IgD | REA740 | Naive B cells | VioBlue® | 450/50 (violet) |
| CD27 | REA499 | Memory B cells | PE-Vio 770 | 750LP (blue) |
| CD38 | REA572 | Plasmablasts/plasma cells | Vio Bright FITC | 525/50 (blue) |
| CD138 | REA929 | Plasma cells | APC | 655-730 (red) |
| CD86 | REA968 | Activation marker | PE | 585/40 (blue) |
| HLA-DR | REA805 | Activation marker | VioGreen™ | 525/50 (violet) |
Gating strategy
View details
Materials
Antibody panel details
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| 7-AAD Staining Solution | - | Viability | - | 655-730 (blue) |
| CD20 | REA780 | B cells | APC-Vio® 770 | 750LP (red) |
| IgD | REA740 | Naive B cells | VioBlue® | 450/50 (violet) |
| CD27 | REA499 | Memory B cells | PE-Vio 770 | 750LP (blue) |
| CD38 | REA572 | Plasmablasts/plasma cells | Vio Bright FITC | 525/50 (blue) |
| CD138 | REA929 | Plasma cells | APC | 655-730 (red) |
| CD86 | REA968 | Activation marker | PE | 585/40 (blue) |
| HLA-DR | REA805 | Activation marker | VioGreen™ | 525/50 (violet) |
See sh
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
- Pan B Cell Isolation Kit, human
- 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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