MBTP 24: Immunophenotyping of innate lymphoid cell 2 (ILC2) subsets from mouse mesentery and small intestine using flow cytometry

Miltenyi Biotec-tested panel 24 (MBTP 24)

This application protocol describes the flow cytometric analysis of ILC2 subsets after dissociation of mesentery and small intestine from healthy C57BL/6 mice. Mouse mesentery and small intestine are easily dissociated using the gentleMACS™ Dissociators to quickly obtain viable single cell suspensions ready for downstream applications, such as flow cytometric analysis.

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Protocol

Staining protocol

FcR blocking

Volumes given below are 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 centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
Cells were resuspended with up to 10⁷ nucleated cells per 90 μL of buffer.
10 μL of FcR Blocking Reagent, mouse was added, mixed well, and incubated for 10 minutes at 4 °C.

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).

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 1 µL of propidium iodide was added right before flow cytometric analysis using the MACSQuant® Analyzer.

Flow cytometric analysis

Gating strategy showing the analysis of ILC2 subsets from mouse mesentery and small intestine. Mesentery and small intestine cells were stained with the previously described panel to analyze ILC2 subsets. Samples were gated on lymphocytes using FSC-A/SSC-A gating (A). Samples were gated on single cells using FSC-W/FSC-H and SSC-W/SSC-H gating within the lymphocyte gate (data not shown). Live lymphocytes were defined as propidium iodide-negative and CD45.2⁺ cells (B). Within lineage-negative cells, ILC2s were identified as CD90.2⁺ Sca-1⁺ KLRG1⁺ cells (C, D). Alternative REAfinity™ Antibodies with similar performance results are available. Please refer to table 2 for more information.

Materials

Reagent and instrument requirements

Antibody panel details

Biotinylated antibodies in this panel are used for lineage definition.

Table 1: Antibody panel

Specificity	Clone	Purpose	Fluorochrome	Detection filter (nm) (laser)
CD3	17A2*	Lineage	Biotin	-
CD4	REA604	Lineage	Biotin	-
CD8a	53-6.7*	Lineage	Biotin	-
CD11c	REA754	Lineage	Biotin	-
FcεRIα	MAR-1*	Lineage	Biotin	-
NK1.1	PK136*	Lineage	Biotin	-
CD19	REA749	Lineage	Biotin	-
Ter-119	REA847	Lineage	Biotin	-
CD5	REA421	Lineage	Biotin	-
F4/80	REA126	Lineage	Biotin	-
Gr-1	RB6-8C5*	Lineage	Biotin	-
Biotin	REA746	-	APC	655-730 (red)
Sca-1	REA422	ILC2	Vio® Bright FITC	525/50 (blue)
KLRG1	2F1*	ILC2	PE	585/40 (blue)
CD90.2	30-H12*	ILC2	PerCP-Vio 770	655-730 (blue)
CD45.2	104-2*	Lymphocytes	APC-Vio 770	750LP (red)
Propidium Iodide Solution	-	Viability	-	655-730 (blue)

*alternative REAfinity™ Antibodies available (refer to table 2)

Table 2: Alternative REAfinity Antibodies

Specificity	Clone	Purpose	Fluorochrome	Detection filter (nm) (laser)
CD3	REA641	Lineage	Biotin	-
CD8a	REA601	Lineage	Biotin	-
FcεRIα	REA1079	Lineage	Biotin	-
NK1.1	REA1162	Lineage	Biotin	-
Gr-1	REA810	Lineage	Biotin	-
KLRG1	REA1016	ILC2	PE	585/40 (blue)
CD90.2	REA1167	ILC2	PerCP-Vio 770	655-730 (blue)
CD45.2	REA1223	Lymphocytes	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
FcR Blocking Reagent, mouse (# 130-092-575)
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 24: Immunophenotyping of innate lymphoid cell 2 (ILC2) subsets from mouse mesentery and small intestine using flow cytometry

Miltenyi Biotec-tested panel 24 (MBTP 24)

Protocol

Materials