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)
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.
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.
Materials
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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