The assessment of viability in isolated rat hepatocytes subjected to cold or subzero non-freezing preservation protocols using a propidium iodide modified test.
A rapid and simple assay (6 min, two steps) is described for determination of cell viability of hepatocytes subjected to cold preservation protocols. In this method, cells are incubated with the fluorescent marker propidium iodide (PI) and the fluorescence intensity is measured before (direct fluorescence--Fd) and after (total fluorescence--Ft) addition of digitonin, which allows the dye to enter the hepatocytes. The Fd originated from non-viable cells that have membrane damage and taken up PI. The Ft originated from all cells in the sample. The ratio between the two fluorescence values is used as an indicator of cell viability. The assay was challenged versus two classical viability tests: LDH retention and Trypan Blue exclusion. Our assay shows good correlation only with Trypan Blue test. In addition, a fluorescence confocal microscopy protocol was used to evaluate the possible toxicity of PI in hepatocyte suspensions.
Giraudi
Pablo J
PJ
Farmacología, Departamento de Ciencias Fisiológicas, Universidad Nacional de Rosario, Argentina.
Almada
Luciana L
LL
Mamprin
María E
ME
Guibert
Edgardo E
EE
Furno
Graciela
G
Rodriguez
Joaquín V
JV
D000818:Animals; D002470:Cell Survival; D003080:Cold Temperature; D015925:Cryopreservation / Q000379:methods*; D005456:Fluorescent Dyes; D022781:Hepatocytes / Q000166:cytology*; D008297:Male; D011419:Propidium; D051381:Rats; D017208:Rats, Wistar
D005456:Fluorescent Dyes; D011419:Propidium