MBTP 20: Immunophenotyping of the activation status of NK cells from human PBMCs using flow cytometry
Miltenyi Biotec-tested panel 20 (MBTP 20)
Miltenyi Biotec-tested panel 20 (MBTP 20)
This application protocol describes the flow cytometric analysis of the activation status of NK cells isolated from PBMCs from whole blood of a healthy donor. Immunophenotyping of NK cells was performed on all PBMCs, directly after isolation via CD3 depletion and CD56 enrichment, and after 14 days of culture.
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 of PEB buffer and and 1 µL of 7-AAD Staining Solution was added right before flow cytometric analysis using the MACSQuant® Analyzer.
Materials
Antibody panel details
| Channel | Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| V1 | CD56 | REA196 | NK cells | Vio® Bright V423 | 450/50 (violet) |
| V2 | CD45 | REA747 | Leukocytes | VioGreen™ | 525/50 (violet) |
| B1 | CD335 (NKp46) | REA808 | Activation marker | Vio Bright B515 | 525/50 (blue) |
| B2 | CD336 (NKp44) | REA1163 | Activation marker | PE | 585/40 (blue) |
| B3 | 7-AAD Staining Solution | - | Viability | - | 655–730 (blue) |
| B3 | CD3 | REA613 | Cell type exclusion | PerCP-Vio 700 | 655–730 (blue) |
| CD14 | REA599 | Cell type exclusion | |||
| B4 | CD337 (NKp30) | REA823 | Activation marker | PE-Vio 770 | 750LP (blue) |
| R1 | CD314 (NKG2D) | REA797 | Activation marker | APC | 655–730 (red) |
| R2 | CD16 | REA423 | Maturation marker | APC-Vio 770 | 750LP (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
- 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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