Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry.
The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome p450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.
Department of Medical Biochemistry, Flinders University, Bedford Park, South Australia 5042, Australia. alan.wigg@flinders.edu.au
Alan J
AJ
Wigg
John W
JW
Phillips
Loretta
L
Wheatland
Michael N
MN
Berry
D000255:Adenosine Triphosphate / Q000032:analysis; D000418:Albumins / Q000032:analysis / Q000096:biosynthesis; D000641:Ammonia / Q000032:analysis / Q000378:metabolism; D000818:Animals; D002452:Cell Count; D002470:Cell Survival; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods*; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000032:analysis / Q000378:metabolism; D019344:Lactic Acid / Q000032:analysis / Q000378:metabolism; D008297:Male; D011419:Propidium / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D014343:Trypan Blue / Q000737:chemistry; D014508:Urea / Q000032:analysis / Q000378:metabolism
D000418:Albumins; D019344:Lactic Acid; D011419:Propidium; D000641:Ammonia; D000255:Adenosine Triphosphate; D014508:Urea; D003577:Cytochrome P-450 Enzyme System; D007770:L-Lactate Dehydrogenase; D014343:Trypan Blue