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.摘要：发育中心脏由多种细胞类型构成，其中主要为心肌细胞(cardiomyocytes)。因此，阐明发育中心脏的调控机制需开展心肌细胞特异性建模研究。斑马鱼(zebrafish)是研究心脏发育的理想模型，因其在发育阶段与人类心脏共享诸多生理特征。本研究提供一套源自斑马鱼幼体的全面的心肌细胞特异性流式细胞术(flow cytometry)数据集。 流式细胞术实验流程： 细胞分选采用BD Influx流式细胞分选仪（BD Bioscience，美国）完成。向细胞悬液中加入终浓度为10 μg/mL的碘化丙啶(Propidium iodide)以剔除死细胞。分选前以绿色荧光蛋白(GFP)阴性细胞对流式细胞分选仪进行校准，以设定自发荧光水平。GFP阳性与GFP阴性细胞均收集于0.125 M甘氨酸-磷酸盐缓冲液中，经液氮速冻后保存于-80℃待用。若开展微阵列分析(microarray analysis)，则省略多聚甲醛(paraformaldehyde, PFA)固定步骤，将细胞分选至含20%胎牛血清的完全L-15 Leibovitz培养基（Gibco）中。 所有分选操作均采用70 µm口径喷嘴，以60 psi的鞘液压力，于室温下进行，将细胞收集至含300 µl RNAlater™ 稳定液（ThermoFisher Scientific，货号AM7020）的低吸附离心管（Sigma，货号Z666505）中。分选门控通过平行处理的同龄野生型幼体的单细胞悬液进行设定。为区分GFP阳性心肌细胞与自发荧光的体细胞（如眼部色素细胞或侧线色素细胞），需设置补偿参数，仅分选GFP阳性且碘化丙啶红色通道无荧光信号的事件。采用野生型TU品系的细胞设定分选门限，使GFP阳性事件的占比最高不超过0.01%。Tg(myl7::GFP)转基因幼体细胞悬液中GFP阳性事件的占比通常约为0.15%。同时包含GFP阳性与GFP阴性事件的液滴（占GFP阳性事件的约20%）将被排除。FACS数据采用FlowJo软件进行分析。 分选得到的细胞经3000×g离心后沉淀，吸弃上清液后，将细胞沉淀经液氮快速冷冻，并保存于-80℃。