A microfluorometric viability assay for isolated human and rat islets of Langerhans.
A microfluorometric viability assay for isolated human and rat islets of Langerhans has been developed using the fluorochromes fluorescein diacetate and propidium iodide. Fluoroscein diacetate causes live cells to fluoresce green under blue light excitation (490 nm); propidium iodide causes dead cells to fluoresce red under green light excitation (545 nm). The fluorescence intensity from the live and dead cells within a single islet was selectively measured by photometry using 520 nm and 610 nm barrier filters with blue and green light excitation respectively. All measurements were corrected for background fluorescence. It was necessary to incubate single islets with the fluorochrome mixture for 105 min in order to achieve maximum fluorescence intensity. It was found that when 50 microliters of a fluorochrome mixture containing 0.67 mumol/l fluorescein diacetate and 4.0 mumol/l propidium iodide was incubated with a single islet and the fluorescence from live (blue light excitation) and dead (green light excitation) cells measured, then the proportion of dead cells within the islet was equal to the (propidium iodide fluorescence)--(0.04 x fluorescein fluorescence) divided by the sum of the (fluorescein fluorescence) and (propidium iodide fluorescence--(0.04 x fluorescein fluorescence]. The proportion of dead cells within single human or rat islets measured by microfluorometry was found to correlate highly significantly (r = 0.99, p less than 0.001) with the proportion of dead cells measured by dissociating the same islet into a single cell suspension and counting the actual proportion of dead cells. This assay therefore provides a rapid, accurate and objective measurement of the proportion of dead cells within isolated human and rat islets.
London
N J
NJ
Department of Surgery, Leicester University, UK.
Contractor
H
H
Lake
S P
SP
Aucott
G C
GC
Bell
P R
PR
James
R F
RF
D000818:Animals; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology* / Q000473:pathology; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D011419:Propidium; D051381:Rats
D005452:Fluoresceins; D005456:Fluorescent Dyes; D011419:Propidium; C018506:diacetylfluorescein