Division of Developmental and Translational Diabetes and Endocrinology Research, Department of Diabetes and Metabolic Researches
Beckman Research Institute of City of Hope,
Newport Green for islet-specific viability assay by semi-automated method
Hirotake Komatsu1, Keiko Omori1, Mounika Parimi1, Jeffrey Rawson1, Fouad Kandeel1, Yoko Mullen1.
1Department of Diabetes and Metabolic Researches, Beckman Research Institute of City of Hope, Duarte, CA, United States
Aim/Background: Islet viability is one of the key factors for the post-transplant islet function and successful islet transplantation. Viability assessment has most commonly used fluorescein diacetate (FDA)/propidium iodide (PI) staining. However, viability results are reported not to correlate to graft function properly. FDA penetrates into all cells in islet samples, including islets and contaminating acinar cells. Furthermore, the manual method for determination of percent viability is highly subjective. Newport Green (NG) is a zinc-specific fluorescent dye, and thus reacts specifically with beta-cells in which zinc is abundant in the cytoplasm. Two kinds of NG dyes, NG-DCF and NG-PDX, are currently available. We examined their characteristics and explored the potential of NG dyes to improve islet viability assessment.
Materials and Methods: ZnSO4 was used to examine the reaction of NG dyes to zinc. To determine the zinc-specific reactivity of the dyes, a beta cell line, NIT-1, a zinc-free pancreatic duct cell line, PANC-1, and human islets were used. We also prepared human islets with different purities by combining islets with non-islet tissue to make the final purity of 30 and 80%. To increase the fluorescent intensity in a shorter incubation time, two different conditions, an addition of F127 and the incubation temperature at 37°C vs. room temperature, were tested. Microscopic photographs of stained cells were taken and analyzed by the cellSense software (OLYMPUS). Images were automatically assembled, so that the entire well area of a 96-well plate can be assessed. This was followed by the analyses of the area measurements. The percent viability was calculated as follows; Viability(%) = 100- [(Area_PI in Area_NG/ Area_NG)* 100].
Results: Fluorescent intensity of NGs positively correlated to the zinc concentration, but FDA did not. NG-DCF reacted specifically to a beta cell line NIT-1, but not to PANC-1. Also, NG-DCF showed higher specificity than NG-PDX as tested with human islets. NG-DCF accurately identified the islet area irrespective of islets purity, while both FDA and NG-PDX did not. Although NG generally requires a longer staining-incubation time to accurately detect zinc-positive cells, the use of poloxamer F127 and incubation at 37°C permitted the evaluation within 30 min. We also introduced a semi-automated measurement of NG-DCF/PI staining in the entire well area of the 96-well plate. Using the assembled image, the PI-positive area was measured only in the NG-DCF-positive area. Since PI-positive dead cells in non-islet tissues were not included, this method enabled us to obtain a true “percent islet viability” objectively and reproducibly.
Conclusions: NG-DCF/PI staining is an easy and reliable islet specific viability assay. With the combination of NG-DCF/PI staining and semi-automated measurement, highly objective islet viability assessments can be achieved.
07:00 - 08:00
|Mini-Orals: Experimental islet transplantation||Newport Green for islet-specific viability assay by semi-automated method||Room 110|
17:30 - 18:30
|Joint Poster Session||Survival of subcutaneous islet transplant in hyperoxia||Main Foyer 1 & 2|