MBTP 37: Immunophenotyping of neonatal cardiomyocytes from mouse using flow cytometry

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.

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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.

Flow cytometry analysis of enriched neonatal mouse CMs. Neonatal mouse CMs were isolated from dissociated whole hearts or individually dissociated chambers using the Neonatal Cardiomyocyte Isolation Kit, mouse. The cells were labeled with Cardiac Troponin T Antibody-FITC, MLC2a Antibody-VioBlue, and MLC2v Antibody-APC after enrichment and analyzed by flow cytometry using the MACSQuant Analyzer 10. CM subtypes were characterized by differential expression of MLC2a (in atrial CMs, A-C) and MLC2v (in ventricular CMs, D-F).

Materials

Reagent and instrument requirements

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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MBTP 37: Immunophenotyping of neonatal cardiomyocytes from mouse using flow cytometry

Miltenyi Biotec-tested panel 37 (MBTP 37)

Protocol

Materials

Protocol

Staining protocol

Intracellular staining

Flow cytometric analysis

Materials

Reagent and instrument requirements

Antibody panel details

Additional reagents used

Trademarks and disclaimers

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About Miltenyi Biotec

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MBTP 37: Immunophenotyping of neonatal cardiomyocytes from mouse using flow cytometry

Miltenyi Biotec-tested panel 37 (MBTP 37)

Protocol

Materials

Protocol

Staining protocol

Intracellular staining

Flow cytometric analysis

Materials

Reagent and instrument requirements

Antibody panel details

Additional reagents used

Trademarks and disclaimers

Shop

Careers

About Miltenyi Biotec