Comprehensive Analysis of Human Pancreatic Islets Using Flow and Laser Scanning Cytometry

摘要: Assessing islet cellular composition and β cell viability using Flow Cytometry (FC) Laser Scanning (LSC) may aid in determining the transplant quality of islets. Human islets (2500 IEQ, n = 44, purity ≥80%) dissociated into a single suspension were stained with ductal marker CA19, Newport Green (NG) FluoZin3 (FL3) for β-cell identification, TMRE to assess mitochondrial membrane potential, DAPI identify live vs. dead cells, Annexin-V/DAPI differentiate apoptotic necrotic cells. For LSC, preparations (n 9) insulin (β-cells), glucagon (α-cells), somatostatin (δ cells), pancreatic polypeptide (ppp cells). Fluorescence microscopy (EtBr/FDA) response also measured. DAPI− staining was 73.78% ± 1.37, while EtBr/FDA 96% 0.48. 52.5% 3.73 all cells NG+, which 58.08% 2.61 NG+/TMRE+. 26) showed 13.8% 0.89 apoptotic, 27.2% 2.0 necrotic, 51.9% 2.22 26.0% 5.19 CA19 positive 17), 45.5% 4.37 TMRE+, 5.2% 1.2 TMRE+ NG+/CA19+. NG FL3 similar 8). Comparison short-term (≤2 days) versus long-term (≥3 culture TMRE+/NG+ averages, albeit lower percentages (36.4% vs 51.9%), higher (19.2% 13.8%) (37.4% 27.2%) long-term, as determined by Annexin-V staining. LSC resulted 54.17% 4.62 β-cells, 33.33% 4.16 α-cells, 8.75% 2.5 δ-cells, 3.75% 0.79 ppp There is no significant difference between (P ≤ .55). FC provide valuable information about quality, could potentially be used evaluating prior transplantation.