Flow cytometry data of zebrafish cardiomyocytes

Abstract: The developing heart is composed of several cell-types, predominantly cardiomyocytes. Therefore, cardiomyocytes-specific modelling is required to understand the regulation of the developing heart. Zebrafish are an ideal model to study heart development, as they share several physiological features with the human heart during development. Here we present a comprehensive, cardiomyocyte-specific flow cytometry dataset from zebrafish larvae. 

Flow cytometry protocol:

Cell sorting was carried out using BD Influx Cell Sorter (BD Bioscience, USA). Propidium iodide (10 µg/mL final concentration) was added to cell suspensions to exclude dead cells. To set the autofluorescence level, Cell Sorter was calibrated with GFP-negative cells before cell separation. GFP-positive and GFP-negative cells were collected in 0.125 M glycine-PBS, frozen in liquid nitrogen and kept at −80°C until use. For microarray analysis, PFA fixation step was omitted, and cells were sorted into complete L-15 Leibovitz medium (Gibco) containing 20% fetal bovine serum.

All sorts were performed with a 70 µm nozzle and 60 psi sheath pressure at room temperature into low-binding tubes (Sigma, #Z666505) containing 300 µl RNAlater™ Stabilization Solution (ThermoFisher Scientific, AM7020). Gates were set using an age-matched single cell suspension of wild type larvae that have been processed in parallel. To discriminate GFP+ cardiomyocytes from autofluorescent body cells (e.g. pigment cells of the eye or lateral line), compensation settings were applied and only events that are GFP+ but show no fluorescence in the red channel for propidium iodide were sorted. Cells from the wild type strain TU were used to assign gate threshold, so that a maximum of 0.01% of the events were considered GFP+. The fraction of GFP+ events in the cell suspension of the Tg(myl7::GFP) transgenic larvae was typically around 0.15%. Droplets that contained both a GFP+ and a GFP- event were excluded that occurred at a frequency of about 20% of GFP+ events. FACS data was analysed with FlowJo software.

The sorted material was pelleted by centrifugation at 3000xg. Supernatant was removed by aspiration, and the pellet was snap-frozen in liquid nitrogen and stored at ‑80˚C.