MBTP 30: Immunophenotyping of KIR-expressing NK cells from human whole blood using flow cytometry
Miltenyi Biotec-tested panel 30 (MBTP 30)
Miltenyi Biotec-tested panel 30 (MBTP 30)
This application protocol describes the analysis of the human KIR repertoire by flow cytometry. An antibody panel is presented that allows the simultaneous assessment of the four major inhibitory KIRs (KIR2DL1, KIR2DL2/DL3/DS2, KIR3DL1) and an additional inhibitory NK cell receptor, namely NKG2A.
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.
- Master mixes for each antibody panel were 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 1 µL of 7-AAD Staining Solution was added right before flow cytometric analysis using the MACSQuant® Analyzer.
Materials
Antibody panel details
Table 1: Antibody panel.
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) | |
| Backbone staining cocktail | 7-AAD Staining Solution | - | Viability | - | 655-730 (blue) |
| CD3 | REA613 | T cells | PerCP-Vio® 770 | 655-730 (blue) | |
| CD14 | REA599 | Monocytes | PerCP-Vio 770 | 655-730 (blue) | |
| CD45 | REA747 | Leukocytes | VioBlue® | 450/50 (violet) | |
| CD56 | REA196 | NK cells | Vio Bright B515 | 525/50 (blue) | |
| Single inhibitory receptor staining cocktail | CD158a (KIR2DL1) | REA284 | KIR2DL1+ NK cells | PE/APC* | 585/40 (blue)/ 655-730 (red) |
| CD158b (KIR2DL2/DL3) | DX27** | KIR2DL2/DL3+ NK cells | PE/APC* | 585/40 (blue)/ 655-730 (red) | |
| CD158e (KIR3DL1) | DX9** | KIR2DL1+ NK cells | PE/APC* | 585/40 (blue)/ 655-730 (red) | |
| CD159a (NKG2A) | REA110 | NKG2A+ NK cells | PE/APC* | 585/40 (blue)/ 655-730 (red) |
Table 2: Complementary antibody cocktails for single inhibitory receptor stainings.
| Inhibitory receptor of interest | CD158a (KIR2DL1) | CD158b (KIR2DL2/DL3) | CD158e (KIR3DL1) | CD159a (NKG2A) |
| Antibody cocktail for single inhibitory receptor staining | CD158a (KIR2DL1)-PE | CD158b (KIR2DL2/DL3)-PE | CD158e (KIR3DL1)-PE | CD159a (NKG2A)-PE |
| - | CD158a (KIR2DL1)-APC | CD158a (KIR2DL1)-APC | CD158a (KIR2DL1)-APC | |
| CD158b (KIR2DL2/DL3)-APC | - | CD158b (KIR2DL2/DL3)-APC | CD158b (KIR2DL2/DL3)-APC | |
| CD158e (KIR3DL1)-APC | CD158e (KIR3DL1)-APC | - | CD158e (KIR3DL1)-APC | |
| CD159a (NKG2A)-APC | CD159a (NKG2A)-APC | CD159a (NKG2A)-APC | - |
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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