MBTP 23: Immunophenotyping of cortical neurons from mouse brain using flow cytometry
Miltenyi Biotec-tested panel 23 (MBTP 23)
Miltenyi Biotec-tested panel 23 (MBTP 23)
This application protocol describes the quantitative analysis of mouse cortical neurons upon isolation and culture using flow cytometry. Mouse brains are easily dissociated using the gentleMACS™ Dissociators to quickly obtain viable single cell suspensions ready for flow cytometric analysis.
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 1 µL of propidium iodide was added right before flow cytometric analysis using the MACSQuant® Analyzer.
Materials
Antibody panel details
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| O4 | REA576 | Oligodendrocytes | PE | 585/40 (blue) |
| CD45 | REA737 | Microglia | VioBlue® | 450/50 (violet) |
| ACSA-2 | IH3-18A3* | Astrocytes | APC | 655-730 (red) |
| CD11b | REA592 | Microglia | Vio® Bright FITC | 525/50 (blue) |
| Propidium Iodide Solution | - | Viability | - | 655-730 (blue) |
* Alternative REAfinity™ Antibody available: ACSA-2 Antibody, anti-mouse, APC, 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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