title: Comprehensive analysis of human pancreatic islets using flow and laser scanning cytometry.
abstract: Assessing islet cellular composition and beta cell viability using Flow Cytometry (FC) and Laser Scanning Cytometry (LSC) may aid in determining the transplant quality of islets. Human islets (2500 IEQ, n = 44, purity >or=80%) dissociated into a single cell suspension were stained with ductal marker CA19, with Newport Green (NG) and FluoZin3 (FL3) for beta-cell identification, with TMRE to assess mitochondrial membrane potential, with DAPI to identify live vs. dead cells, and with Annexin-V/DAPI to differentiate apoptotic and necrotic cells. For LSC, cell preparations (n = 9) were stained for insulin (beta-cells), glucagon (alpha-cells), somatostatin (delta cells), and pancreatic polypeptide (ppp cells). Fluorescence microscopy (EtBr/FDA) and insulin response were also measured. DAPI- staining was 73.78% +/- 1.37, while EtBr/FDA was 96% +/- 0.48. 52.5% +/- 3.73 of all cells were NG+, of which 58.08% +/- 2.61 were NG+/TMRE+. Annexin-V/DAPI staining (n = 26) showed 13.8% +/- 0.89 apoptotic, 27.2% +/- 2.0 necrotic, and 51.9% +/- 2.22 live cells. 26.0% +/- 5.19 of cells were CA19 positive (n = 17), of which 45.5% +/- 4.37 were also TMRE+, and 5.2% +/- 1.2 of the TMRE+ were also NG+/CA19+. NG and FL3 showed similar staining (n = 8). Comparison of short-term (<or=2 days) versus long-term (>or=3 days) culture showed similar TMRE+/NG+ averages, albeit lower percentages of live (36.4% vs 51.9%), and higher percentages of apoptotic (19.2% vs 13.8%) and necrotic cells (37.4% vs 27.2%) for long-term, as determined by Annexin-V staining. LSC resulted in 54.17% +/- 4.62 beta-cells, 33.33% +/- 4.16 alpha-cells, 8.75% +/- 2.5 delta-cells, and 3.75% +/- 0.79 ppp cells. There is no significant difference between insulin positive cells and NG positive cells (P <or= .55). FC and LSC provide valuable information about islet quality, which could potentially be used for evaluating islets prior to transplantation.
authors: Southern California Islet Cell Resources Center, Department of Diabetes, Endocrinology and Metabolism, Beckman Research Institute of City of Hope, Duarte, California 91010, USA.
authors: I
authors: I
authors: Iglesias
authors: K
authors: K
authors: Bentsi-Barnes
authors: C
authors: C
authors: Umeadi
authors: L
authors: L
authors: Brown
authors: F
authors: F
authors: Kandeel
authors: I H
authors: IH
authors: Al-Abdullah
mesh_terms: D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival; D004396:Coloring Agents; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology; D007515:Islets of Langerhans / Q000166:cytology*; D047569:Laser Scanning Cytometry / Q000379:methods*
chemical_list: D004396:Coloring Agents