MBTP 30: Immunophenotyping of KIR-expressing NK cells from human whole blood using flow cytometry

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.

Print protocol

Protocol

Staining 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.

Flow cytometric analysis

Gating strategy showing the analysis of NK cells expressing single inhibitory KIRs from human whole blood. NK cells from human whole blood were stained as described before. Lymphocytes were gated based on CD45 expression (A). Dead cells, T cells, monocytes, and debris were excluded by gating on 7-AAD^- CD3^- CD14^- cells (B) and based on FSC-A/SSC-A gating (C). Expression of single inhibitory KIRs by CD56⁺ NK cells (D) was assessed by analysis of KIR2DL1 (E) or KIR2DL2 (F) or KIR3DL1 (G), or NKG2A (H) expression.

Materials

Reagent and instrument requirements

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)

* The signal for the inhibitory receptor of interest is detected in the PE channel of the flow cytometer, whereas the other inhibitory receptors are detected collectively in the APC channel.

** alternative REAfinity™ Antibodies available (clones REA1006 and REA1005)

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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MBTP 30: Immunophenotyping of KIR-expressing NK cells from human whole blood using flow cytometry

Miltenyi Biotec-tested panel 30 (MBTP 30)

Protocol

Materials