Flow Cytometry Analysis of Myocytes

Fixed ventricular cells in suspension were treated with DNAse-free RNAse A 2μg/ml (Sigma, St. Louis, MO) and labeled with propidium iodide (PI, BD Pharmingen) prior to flow cytometry to detect nucleated particles as described[6 (link), 19 (link), 20 (link)]. Cells from pellet, supernatant (i.e. post-spin) and parent suspension (i.e. pre-spin) were analyzed for light-scatter signatures using a standard FACScan (BD Biosciences, San Jose, CA) with a standard 430μm x 180μm flow cell as described.[6 (link), 19 (link), 20 (link)] Side light-scatter is validated as a size indicator of large myocytes.[6 (link)]
Myocytes were labeled with mouse anti-beta-myosin heavy chain (β-MyHC) IgG (NOQ7.5.4D, Sigma-Aldrich, St Louis, MO) using a Zenon kit (Invitrogen) conjugated to Alexa 488 (green) following manufacture’s recommendations. The NOQ7.5.4 has been previously validated to identify fetal mouse myocytes and adult rabbit myocytes expressing the β-isoform (MHY7) of MyHC.[6 (link)]

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López J.E., Jaradeh K., Silva E., Aminololama-Shakeri S, & Simpson P.C. (2017). A method to increase reproducibility in adult ventricular myocyte sizing and flow cytometry: Avoiding cell size bias in single cell preparations. PLoS ONE, 12(10), e0186792.

Publication 2017

DNAse-free RNAse A propidium iodide (PI, FACScan Zenon kit

Adult Cell Dnase Fetal Flow cytometry Isoform Light Mouse Myocytes Myosin heavy chain Parent Propidium iodide Rabbit Rnase Ventricular X flow

Corresponding Organization : San Francisco VA Medical Center

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Variable analysis

independent variables

Treatment with DNAse-free RNAse A 2μg/ml

dependent variables

Light-scatter signatures (size indicator of large myocytes) of cells from pellet, supernatant, and parent suspension
Myocyte labeling with mouse anti-beta-myosin heavy chain (β-MyHC) IgG

control variables

Fixed ventricular cells in suspension
Labeling with propidium iodide (PI)
Flow cytometry using a standard FACScan with a 430μm x 180μm flow cell

positive controls

Previously validated that the NOQ7.5.4 antibody identifies fetal mouse myocytes and adult rabbit myocytes expressing the β-isoform (MHY7) of MyHC

negative controls

No negative controls explicitly mentioned

Annotations

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