Journal: PLoS ONE

Article Title: Cryopreservation and post-thaw characterization of dissociated human islet cells

doi: 10.1371/journal.pone.0263005

Figure Lengend Snippet: (A) Representative FACS plots showing cell viability and the distribution of distinct pancreatic single islet cell populations. Islets (from donor ID# R292) were dispersed as described in the Materials and Methods, and analyzed before cryopreservation (fresh, unfrozen control). Dispersed islet cells from the same donor were cooled at 1°C/min to –40°C in the presence of 10% DMSO and 6% HES, then stored in liquid nitrogen. Cells were thawed rapidly in a 37°C water bath, and the cryoprotectants were removed by serial dilution as described in the Materials and Methods. The cells were analyzed immediately ( after w/o culture) or after overnight culture ( after w culture). The cells were first stained with LIVE/DEAD Fixable near-IR dead cell dye to assess cell viability and analyzed using specific cell surface markers to stain distinct cell populations: CD31 for endothelial cells; CD45 for leukocytes; CD90 for mesenchymal cells; EpCAM for epithelial cells including acinar, ductal and islet cells; HPx1 for acinar cells and HPi2 for islet cells. FACS analyses were able to detect most of cell populations before after cryopreservation. (B) Summary of FACS analysis of viability and EpCAM (surface marker for islet cells) expression in three pancreatic islet donors. Fresh (black bars) and cryopreserved (red bars) dispersed islet cells from donors (see ) were analyzed by flow cytometry. Cryopreserved cells were also cultured overnight and analyzed the next day (blue bars). Left panel: Cell viability is expressed as mean relative abundance ± standard error of the mean with actual data points represented as individual dots. Right panel: EpCAM expression.

Article Snippet: The following antibodies were used for FACS experiments at 1:100 (v/v) final concentration, CD31-FITC (BioLegend, 303103, San Diego, CA), CD45-PE/Dazzle594 (BioLegend, 304052), CD90-Alexa Fluor 700 (BioLegend, 328120), EpCAM-APC (BioLegend, 324208), HPi2-Alexa405 (Novus Biologicals, NBP1-18946AF405, Centennial, CO), and HPx1-PE (Novus Biologicals, NBP1-18951PE).

Techniques: Control, Serial Dilution, Staining, Marker, Expressing, Flow Cytometry, Cell Culture

Journal: Cell metabolism

Article Title: Patch-seq links single-cell transcriptomes to human islet dysfunction in diabetes

doi: 10.1016/j.cmet.2020.04.005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: HPx1-Alexa-647 , Novus Biologicals , NBP1-18951AF647.

Techniques: Control, SYBR Green Assay, Software