MBTP 8: Immunophenotyping of NK cells from human whole blood using flow cytometry

Miltenyi Biotec-tested panel 8 (MBTP 8)

NK cells were isolated from whole blood of a healthy donor and analyzed for cell subtypes.


This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.

Gating strategy showing the analysis of NK cell subsets from whole human blood. NK cells subsets from human whole blood were stained using the previously described panel. Cells were gated on lymphocytes and doublets and debris were excluded using FSC/SSC gating (data not shown). NK cell subsets were further defined as CD56bright (CD56bright CD16 ) and CD56dim (CD56dim CD16+) (data not shown). KIR2D, CD314 (NKG2D), CD159a (NKG2A), and CD159c (NKG2C) expression by CD56dim NK cells (A-D) and CD56bright NK cells (E-H) was analyzed. Overlay with isotype controls (red).


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

SpecificityClonePurposeFluorochromeDetection filter (nm) (laser)
CD56REA196NK cellsAPC-Vio® 770750LP (red)
KIR2DREA1042NK cell receptorFITC525/50 (blue)
CD159a (NKG2A)REA110NK cell receptorPE-Vio 770750LP (blue)
CD159c (NKG2C)REA205NK cell receptorPE585/40 (blue)
CD314 (NKG2D)REA797NK cell receptorPE-Vio 615655–730 (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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