MBTP 37: Immunophenotyping of neonatal cardiomyocytes from mouse using flow cytometry
Miltenyi Biotec-tested panel 37 (MBTP 37)
Miltenyi Biotec-tested panel 37 (MBTP 37)
This application protocol describes the characterization of distinct neonatal cardiomyocyte (CM) subtypes (atrial and ventricular) from mouse using flow cytometry.
Protocol
Intracellular staining
- Volumes given below are 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 5 minutes. Supernatant was aspirated completely.
- Cells were resuspended in 100 μL of PBS.
- 100 μL of Inside Fix solution from the Inside Stain Kit was added.
- Cells were mixed and incubated for 10 minutes in the dark at room temperature.
- Cells were washed by addition of 1 mL of PEB buffer and centrifuged at 300×g for 5 minutes. Supernatant was aspirated completely.
- Cells were resuspended in Inside Perm and the following staining antibodies were added according to manufacturer’s recommendations: Cardiac Troponin T Antibody, anti-mouse, FITC, MLC2a Antibody, VioBlue, and MLC2v Antibody, APC.
- Cells were mixed and incubated for 10 minutes in the dark at room temperature.
- Cells were washed with 1 mL of PEB buffer and centrifuged at 300×g for 5 minutes.
- Supernatant was aspirated completely and the pellet was resuspended in 250 μL of PEB buffer.
- Flow cytometric analysis was performed using the MACSQuant Analyzer.
Materials
Antibody panel details
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| Cardiac Troponin T | REA400 | Cardiomyocytes | FITC | 525/50 (blue) |
| MLC2a | REA398 | Cardiomyocyte subtype | VioBlue® | 450/50 (violet) |
| MLC2v | REA401 | Cardiomyocyte subtype | APC | 655-730 (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
- Inside Stain Kit (# 130-090-477)
- Flow cytometer, for example, MACSQuant® Analyzer 10 (# 130-096-343)
- (Optional) MACSQuant Calibration Beads (# 130-093-607)
- (Optional) MACS® Comp Bead Kit, anti-REA (# 130-104-693)
- (Optional) Neonatal Cardiomyocyte Isolation Kit, mouse (# 130-100-825)
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REA400
ICFC, MICS, IF, IHC
EUR 278,00
