Single cell sorting - a survey of practices.

Since its founding, the mission of the Flow Cytometry Research Group (FCRG) has been to provide the flow cytometry community with practical information on fluorescent activated cell sorting that they can implement to better serve their users. The group's current initiative is focused on single cell sorting. Requests to sort one cell per well have increased dramatically in most shared resource laboratories (SRLs) over the last few years, as more researchers are interested in performing cell analysis at the single cell level using techniques such as RNA sequencing (RNAseq). The initial phase of this study was to survey the flow cytometry community to determine what variables (i.e. sample preparation, cell type, sample concentration, environmental factors, etc.) users consider essential for efficient and accurate single cell sorting. The survey was distributed to the flow cytometry community through the Purdue flow cytometry list and through International Society for Advancement of Cytometry (ISAC), American Association for the Advancement of Science (AAAS) and ABRF community messaging boards. Results from the survey showed that the majority of responder's sort into 96 well plates containing 51 -100 ul of tissue culture media. Survey responders ranked the quality of sample preparation, sorting speed, cell concentration, nozzle/pressure, cell type and masking as most important to obtaining a high-quality single cell sort. Interestingly, most survey responders ranked environmental factors such as room temperature or humidity as not critical for a good single cell sort. Based on the survey results, the FCRG will develop detailed protocol(s) for single cell deposition and test the variables that survey responders indicated were most important.