MBTP 8: Immunophenotyping of NK cells from human whole blood using flow cytometry
Miltenyi Biotec-tested panel 8 (MBTP 8)
Miltenyi Biotec-tested panel 8 (MBTP 8)
NK cells were isolated from whole blood of a healthy donor and analyzed for cell subtypes.
Protocol
This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.
- 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).
- Erythrocytes were lysed using Red Blood Cell Lysis Solution.
- Cells were washed by addition of 1 mL of PEB buffer and centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
- 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 10 minutes. Supernatant was aspirated completely.
- Cells were resuspended in 100 µL of PEB buffer and flow cytometric analysis was performed using the MACSQuant® Analyzer.
Materials
The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.
Antibody panel details
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| CD56 | REA196 | NK cells | APC-Vio® 770 | 750LP (red) |
| KIR2D | REA1042 | NK cell receptor | FITC | 525/50 (blue) |
| CD159a (NKG2A) | REA110 | NK cell receptor | PE-Vio 770 | 750LP (blue) |
| CD159c (NKG2C) | REA205 | NK cell receptor | PE | 585/40 (blue) |
| CD314 (NKG2D) | REA797 | NK cell receptor | PE-Vio 615 | 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
- Red Blood Cell Lysis Solution (# 130-094-183)
- 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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REA110
FC
AUD 423.00
REA797
FC
AUD 423.00
REA196
FC
AUD 408.00
