MBTP 20: Immunophenotyping of the activation status of NK cells from human PBMCs using flow cytometry

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.

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.
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.

Flow cytometric analysis

Flow cytometric analysis of the activation status of NK cells within PBMCs, freshly isolated NK cells, and cultured NK cells. PBMCs, freshly isolated NK cells, and isolated NK cells cultured for 14 days were stained using the previously described antibody panel. Samples were initially gated on single cells using FSC-H/FSC-A (data not shown). Live CD45⁺ CD3^– CD14^– cells (A) were further analyzed for expression of CD56 (B) and different activation markers. Compared to NK cells within PBMCs and freshly isolated NK cells, NK cells cultured for 14 days demonstrated an increased expression of CD335 (B), CD336 (C), CD337 (D), and CD314 (F). In contrast, CD16 expression remained unchanged (E).

Materials

Reagent and instrument requirements

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)
B3	CD14	REA599	Cell type exclusion	PerCP-Vio 700	655–730 (blue)
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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MBTP 20: Immunophenotyping of the activation status of NK cells from human PBMCs using flow cytometry

Miltenyi Biotec-tested panel 20 (MBTP 20)

Protocol

Materials