After 24?hours, wells were scored for the presence of a single cell and counted each day to track the clonal growth of individual cells. 7AADdim fraction, suggesting that even low levels of 7AAD staining are indicative of less healthy cell populations. These data indicate that when used in combination with single-cell functional assays, index sorting is a powerful tool for refining cell isolation strategies. This approach can be broadly applied to other single-cell systems, both to improve isolation and to acquire additional cell surface marker information. Introduction Heterogeneity in cell populations poses a significant challenge to understanding the biology of normal and malignant single cells . Advanced multiparameter cell sorting has enabled the isolation of rare subpopulations with properties distinct from those of bulk cell populations, but the vast majority of such populations remain at purities less than 50%, with many fractions substantially lower. This means that when cells are studied at a single-cell level for expression of genes or proteins or are assessed for their functional activity, the majority of the cells assessed are not actually the cells of interest. Therefore, techniques are required either to obtain near-pure cell fractions or to associate individual cells with multiple individual outcomes. The latter is particularly complicated because the majority of such techniques (e.g., gene expression) destroy the cell of interest, making it impossible to assess in a functional assay. Stem cells are generally rare cell populations, and cell number is typically limited in adult mammalian systems , often yielding just a few hundred cells in a single experiment. For example, functional mouse blood stem cells are present at a frequency of 0.004% in the bone marrow and orders of magnitude less in the peripheral blood . Performing large numbers of functional screens using different combinations of multiple cell surface markers is virtually impossible because stem cell transplantation is Cetrorelix Acetate required to validate stem cell function. Efforts have therefore been restricted to adding or subtracting one marker at a time , and virtually no studies have assessed the impact of different levels of expression across multiple markers. Single-cell sorting is a powerful tool in biomedical research as it allows separation and analysis of individual cells. New instrument developments have improved the index sorting function of several commercial cell sorters, making it possible to review the complete flow phenotype of every single cell sorted into a 96-or 384-well plate [5,6]. This technique has already been used to analyze gene expression in planarian stem cells  and the diversity of antibody repertoires in a high-throughput manner [5,6], and most recently we Sav1 have reported its application to stem cell populations . Here we report the use of index sorting in rare mouse hematopoietic stem cell populations as a method to survey multiple different combinations of cell surface marker intensities to resolve subpopulations in cell fractions and to improve purities of functional outcomes. By linking functional in?vitro readouts that associate with stem cell activity to individual single-cell surface marker profiles, we are able to identify contaminating nonfunctional cell fractions and determine the functional importance of higher or lower levels of the stem cell markers EPCR and CD150. Methods BD Influx setup and preparation of plate holder All cell sorting experiments were performed on a BD Influx cell sorter running BD FACS Sortware. Laser alignment was performed using eight-peak rainbow beads (Spherotech), and drop delay was determined using BD Accudrop beads. The plate holder apparatus on a BD Influx does not hold a nonskirted 96-well PCR plate tightly. To create a fitting holder, a 96-well polycarbonate rack typically used to hold individual 1.4-mL polypropylene round-bottom tubes was used. By removing the legs of the rack and shaving the bottom surface to be flat, we were able to create a rigid fit in the sort tray of the Cetrorelix Acetate Influx sorter. Standard Cetrorelix Acetate 96-well PCR plates were able to fit easily into the rack and were secured using individual portions of a pressure-sensitive adhesive (e.g., Blu-Tack) in several locations within the rack. To establish the alignment of the sort plate on the sort stage we performed sorts of 10 beads onto the lid of a 96-well plate. Cells were then index sorted into wells of a 96-well plate and analyzed further. To determine the precision of the cell sorter, cells were index sorted into 96-well PCR plates to execute Fluidigm real-time PCR evaluation (Fig.?1C). Open up in another window Figure?1 Adjustment of BD Influx 96-very well dish workflow and holder of index sorting and analysis. (A) Inserting.