Immunophenotyping of NK cells from mouse spleen using flow cytometry

Miltenyi Biotec-tested panel 18 (MBTP 18)

This application protocol describes the flow cytometric analysis of NK cells after spleen dissociation from healthy C57BL/6 mice. Mouse spleens are easily dissociated using the gentleMACS™ Dissociators to quickly obtain viable single cell suspensions ready for flow cytometric analysis.

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Protocol

Staining protocol

Labeling of cells with Viobility™ Fixable Dye

Viobility Fixable Dye was reconstituted in advance. The vial was warmed up to room temperature to avoid water condensation. One vial of lyophilized Viobility Fixable Dye was reconstituted by addition of 100 µL anhydrous DMSO to the dye vial and mixed until completely dissolved. The solution was aliquoted and stored at –20 °C under anhydrous (desiccant) conditions and protected from light for up to one month.
Volumes given below were for up to 10⁷ nucleated cells. When working with fewer than 10⁷ 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 2×10⁷ nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).

Cell number was determined.
Cells were washed by addition of 1 mL of 1× PBS and centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
Cells were resuspended in 100 µL of 1× PBS.
1 µL of Viobility Fixable Dye was added.
Samples were mixed well and incubated for 15 minutes in the dark at room temperature.
▲Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times.
Cells were washed by addition 1 mL PEB buffer and centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
Step 6 was repeated.
Surface staining was performed.

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 flow cytometric analysis was performed using the MACSQuant® Analyzer.

Flow cytometric analysis

Gating strategy showing the analysis of NK cell maturational stages from mouse spleen. Splenocytes from C57BL/6 mice were stained using the previously described NK cell panel to analyze NK cell maturation stages. Samples were initially gated on single cells using FSC-H/FSC-A gating and on live cells using Viobility-negative gating (data not shown). Mouse NK cells are identified within the CD45⁺/Lineage (CD3/CD19)-negative population (A) as NK1.1⁺ and CD335⁺ cells (B). Mature NK cells express KLRG1 (C). NK cells can further be divided into four maturation stages based upon the expression of CD27 and CD11b (D). Stages in order from less to more mature are: S1<S2<S3<S4.

Materials

Reagent and instrument requirements

Antibody panel details

Specificity	Clone	Purpose	Fluorochrome	Detection filter (nm) (laser)
CD11b	REA592	NK cell maturation	VioBlue®	450/50 (violet)
Viobility™ 405/520 Fixable Dye	-	Viability	-	525/50 (violet)
CD335 (NKp46)	REA815	NK cells	FITC	525/50 (blue)
KLRG1	REA1016	Mature NK cells	PE	585/40 (blue)
CD3e	REA606	Cell type exclusion	PE-Vio® 615	655-730 (blue)
CD19	REA749	Cell type exclusion	PE-Vio 615	655-730 (blue)
NK1.1	PK136*	NK cells	PE-Vio 770	750LP (blue)
CD27	REA499	NK cell maturation	APC	655-730 (red)
CD45	REA737	Leukocytes	APC-Vio 770	750LP (red)

*alternative REAfinity™ Antibody available: NK1.1 Antibody, anti-mouse, PE-Vio 770, REAfinity

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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Immunophenotyping of NK cells from mouse spleen using flow cytometry

Miltenyi Biotec-tested panel 18 (MBTP 18)

Protocol

Materials

Protocol

Staining protocol

Labeling of cells with Viobility™ Fixable Dye

Surface staining

Flow cytometric analysis

Materials

Reagent and instrument requirements

Antibody panel details

Additional reagents used

Trademarks and disclaimers

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Immunophenotyping of NK cells from mouse spleen using flow cytometry

Miltenyi Biotec-tested panel 18 (MBTP 18)

Protocol

Materials

Protocol

Staining protocol

Labeling of cells with Viobility™ Fixable Dye

Surface staining

Flow cytometric analysis

Materials

Reagent and instrument requirements

Antibody panel details

Additional reagents used

Trademarks and disclaimers

Shop

Careers

About Miltenyi Biotec