Enumeration of islets by nuclei counting and light microscopic analysis
| Autor: | Michael J. Rappel, Klearchos K. Papas, Gordon C. Weir, Daryl E. Powers, Susan Bonner-Weir, Anna Pisania, Abdulkadir Omer, Clark K. Colton |
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| Přispěvatelé: | Massachusetts Institute of Technology. Department of Chemical Engineering, Pisania, Anna, Papas, Klearchos K., Powers, Daryl E., Rappel, Michael J., Colton, Clark K. |
| Rok vydání: | 2010 |
| Předmět: |
endocrine system
Pathology medicine.medical_specialty endocrine system diseases Coefficient of variation Cell Count 030209 endocrinology & metabolism Biology islet enumeration Article Pathology and Forensic Medicine Islets of Langerhans Mice 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Hemocytometer Cell Line Tumor Lysis buffer Microscopy medicine Enumeration Animals Humans Molecular Biology 030304 developmental biology Cell Nucleus 0303 health sciences geography geography.geographical_feature_category Chromatography DNA Cell Biology Islet Rats Staining chemistry DTZ staining Dithizone Islets nuclei counting light microscopy |
| Zdroj: | Laboratory investigation; a journal of technical methods and pathology PMC |
| ISSN: | 0023-6837 |
| DOI: | 10.1038/labinvest.2010.125 |
| Popis: | Author Manuscript 2011 May 1. Islet enumeration in impure preparations by conventional dithizone staining and visual counting is inaccurate and operator dependent. We examined nuclei counting for measuring the total number of cells in islet preparations, and we combined it with morphological analysis by light microscopy (LM) for estimating the volume fraction of islets in impure preparations. Cells and islets were disrupted with lysis solution and shear, and accuracy of counting successively diluted nuclei suspensions was verified with (1) visual counting in a hemocytometer after staining with crystal violet, and automatic counting by (2) aperture electrical resistance measurement and (3) flow cytometer measurement after staining with 7-aminoactinomycin-D. DNA content averaged 6.5 and 6.9 pg of DNA per cell for rat and human islets, respectively, in agreement with literature estimates. With pure rat islet preparations, precision improved with increasing counts, and samples with about greater than or equal to 160 islets provided a coefficient of variation of about 6%. Aliquots of human islet preparations were processed for LM analysis by stereological point counting. Total nuclei counts and islet volume fraction from LM analysis were combined to obtain the number of islet equivalents (IEs). Total number of IE by the standard method of dithizone staining/manual counting was overestimated by about 90% compared with LM/nuclei counting for 12 freshly isolated human islet research preparations. Nuclei counting combined with islet volume fraction measurements from LM is a novel method for achieving accurate islet enumeration. National Institutes of Health (U.S.) (Grant NCRR ICR U4Z 16606) National Institutes of Health (U.S.) (Grant R01-DK063108-01A1) National Institutes of Health (U.S.) (Grant NCRR ICR U42 RR0023244-01) Joslin Diabetes and Endocrinology Research Center (Grant DK36836) Diabetes Research & Wellness Foundation Juvenile Diabetes Research Foundation International (Islet Transplantation, Harvard Medical School ) |
| Databáze: | OpenAIRE |
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