MBTP 39: Immunophenotyping of plasma cells from human multiple myeloma bone marrow using flow cytometry
Miltenyi Biotec-tested panel 39 (MBTP 39)
Miltenyi Biotec-tested panel 39 (MBTP 39)
This application protocol describes the analysis of plasma cells from human multiple myeloma bone marrow by flow cytometry.
Protocol
Surface staining
- Volumes given below are 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.
- Cells were resuspended in buffer and the following staining antibodies were added according to manufacturer’s recommendations: CD138 Antibody, anti-human, APC, CD38 Antibody, anti-human, Vio Bright FITC, REAfinity™, CD235a Antibody, anti-human, PE-Vio 770, REAfinity, CD45 Antibody, anti-human, VioGreen, REAfinity, CD19 Antibody, anti-human, VioBlue, REAfinity, CD28 Antibody, anti-human, APC-Vio 770, REAfinity, and CD56 Antibody, anti-human, APC-Vio 770, REAfinity.
- Cells were mixed and incubated for 10 minutes in the dark at 4 °C.
- Cells were washed by addition of 1–2 mL of PEB buffer and centrifuged at 300×g for 10 minutes at 4 °C. Supernatant was aspirated completely.
- Cells were resuspended in 100 µL of buffer before flow cytometric analysis using the MACSQuant® Analyzer.
View details
Materials
Antibody panel details
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| CD138 | 44F9 | Plasma cells (PCs) | APC | 655–730 (red) |
| CD38 | REA671 | PCs | Vio® Bright FITC | 525/50 (blue) |
| CD235a | REA175 | RBC | PE-Vio 770 | 750LP (blue) |
| CD45 | REA747 | Erythroblast exclusion | VioGreen™ | 525/50 (violet) |
| CD19 | REA675 | Myeloma PCs | VioBlue® | 450/50 (violet) |
| CD28 | REA612 | PCs | APC-Vio 770 | 750 LP (red) |
| CD56 | REA196 | Myeloma PCs | APC-Vio 770 | 750 LP (red) |
| Propidium iodide | - | Viability | - | 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
- Flow cytometer, for example, MACSQuant® Analyzer 10 (# 130-096-343)
- (Optional) MACS® Comp Bead Kit, anti-REA (# 130-104-693)
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REA196
FC
USD 370.00
REA612
FC
USD 370.00
REA671
FC
USD 405.00
REA175
FC
USD 355.00
