title: A rapid method to verify single-cell deposition setup for cell sorters.
abstract: Studying single cells reveals biology that cannot be explored using bulk techniques. Cell sorters provide the opportunity of separating single cells either for cell culture or for downstream molecular applications such as qPCR to study specific gene expression and single cell mRNA sequencing. Some of these molecular studies can be expensive so the investigator will often want reassurance that the cell sorter can reliably deposit a single droplet into each well of a 96-well or 384-well plate. Such plates may contain very small volumes of fluid as reducing the volume of fluid used can reduce the cost of the assay. To miss some of the wells could leave the data set incomplete requiring costly repetition. To verify this by microscopy is at best very time consuming and at worst impossible. Here, an inexpensive colorimetric method is described for verifying whether a well, in either a 96- or 384-well plate, did receive a single sorted droplet from a cell sorter into the fluid at the bottom of the well. The droplet consists of particles suspended in an enzymatic solution, horseradish peroxidase, which is deposited into microtiter plate wells containing a substrate, 3,3',5,5'-tetramethylbenzidine. This method requires no special equipment or expertise and is rapid enough to be performed directly prior to the single-cell sorting experiment. © 2016 International Society for Advancement of Cytometry.
authors.affiliation: MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, EH16 4UU, United Kingdom.
authors.forename: Olivia Roos
authors.initials: OR
authors.lastname: Rodrigues
authors.affiliation: The Cytometry Laboratory, Walter and Eliza Hall Institute, Parkville, 3052, Victoria, Australia.
authors.forename: Simon
authors.initials: S
authors.lastname: Monard
mesh_terms: D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology; D001560:Benzidines / Q000737:chemistry; D045744:Cell Line, Tumor; D002469:Cell Separation / Q000592:standards*; D003124:Colorimetry / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D005434:Flow Cytometry / Q000592:standards*; D006735:Horseradish Peroxidase / Q000737:chemistry; D006801:Humans; D007834:Lasers; D011786:Quality Control; D059010:Single-Cell Analysis / Q000295:instrumentation / Q000379:methods*
chemical_list: D001560:Benzidines; C021758:3,3',5,5'-tetramethylbenzidine; D006735:Horseradish Peroxidase
keywords: colorimetric method of detection; flow cytometry; horseradish peroxidase; polystyrene microspheres; single-cell sorting