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Updated: Jul 6, 2026
Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry
Published on: January 28, 2017
Comprehensive analysis of human pancreatic islets using flow and laser scanning cytometry
I Iglesias1, K Bentsi-Barnes, C Umeadi
1Southern California Islet Cell Resources Center, Department of Diabetes, Endocrinology and Metabolism, Beckman Research Institute of City of Hope, Duarte, California 91010, USA.
Insights
Flow cytometry and laser scanning cytometry assess islet cell composition and beta cell viability, aiding in transplant quality evaluation. These methods offer valuable insights for pre-transplant islet assessment.
Area of Science:
- Cell Biology
- Immunology
- Transplantation Science
Background:
- Assessing the quality of pancreatic islets is crucial for successful transplantation.
- Flow Cytometry (FC) and Laser Scanning Cytometry (LSC) are advanced techniques for cellular analysis.
Purpose of the Study:
- To evaluate the utility of FC and LSC in assessing human islet cellular composition and beta cell viability.
- To determine if these methods can aid in predicting islet transplant quality.
Main Methods:
- Human islets were dissociated and stained for various markers including ductal (CA19), beta-cell (Newport Green, FluoZin3, insulin), mitochondrial potential (TMRE), and cell death (DAPI, Annexin-V).
- Laser Scanning Cytometry (LSC) was used to identify different islet endocrine cell types (alpha, beta, delta, PP).
- Fluorescence microscopy and insulin response assays were also performed for comparison.
Main Results:
- FC and LSC successfully differentiated various islet cell populations and assessed viability.
- TMRE staining indicated mitochondrial membrane potential in beta cells, with similar results in short-term and long-term cultures.
- Annexin-V/DAPI staining revealed percentages of apoptotic, necrotic, and live cells, with higher cell death observed in long-term cultures.
Conclusions:
- FC and LSC provide valuable quantitative data on islet cellular composition and viability.
- These cytometric techniques show potential for evaluating islet quality prior to transplantation.
- Further studies can refine these methods for routine pre-transplant islet assessment.
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 (
