Trypan blue hepatocyte viability

Isolated hepatocytes incubated with ionic iron rapidly undergo lipid peroxidation. Some studies have not shown a consequent decrease in viability (as indicated by uptake of trypan blue or release of enzymes). This is probably a result of short incubation times, as changes in viability lag behind increases in lipid peroxidation, and may not occur for more than 2 h after lipid peroxidation begins (Bacon and Britton, 1990). Recent studies have shown strong correlations between increased lipid peroxidation [production of thiobarbituric acid (TBA) reactants] and loss of cell viability (trypan blue staining) (Bacon and Britton, 1989). The significance of the lag between lipid peroxidation and decreases in cell viability is as yet uncertain. 

Viability of the hepatocytes is usually determined by trypan blue exclusion rate. Viability over incubation time can be determined by LDH retention or albumin secretion (Gebhardt 2003). For metabolism... 

C. Hepatocytes are counted in a hemocytometer and viability is determined using the trypan blue exclusion method. Viability is always > 90 % with a yield of 2-3 x 10s cells. Cells are resuspended in medium and diluted to a final concteration of 1 x 106 cells/ml. 

Determine the viability of the hepatocytes using 0.2% trypan blue in saline (Note 5). 

Cryopreserved human hepatocytes from three male and two female donors or freshly isolated male rat hepatocytes are analyzed for viabilities (75-85%) using the trypan blue exclusion methods. Incubations are performed by suspending the hepatocytes in Krebs-biocarbonate buffer followed by addition of a H-labeled compound in methanol. The specific radioactivity of the compounds is 100 Ci/ mol. The final concentration of test compound in the suspension is 10 xM in a final volume of 1 mL (1x10 cells/mL), and the final concentration of methanol does not exceed 0.2% (v/v). Incubations are allowed to proceed at 37 °C for 1 h, and are quenched with acetonitrile (5 mL). The remaining procedures are the same as described in Section 14.2.2 (the protocol for in vitro covalent protein binding in human or rat liver microsomes a test-tube method). Covalent protein binding values in pmol-equiv./mg protein are estimated based on the residual radioactivity in the protein pellets. 

Rat Hepatocytes Rat hepatocytes are freshly isolated by perfusion and are diluted with Krebs-bicarbonate buffer (pH 7.4) to 1 x 10 cells/mL. Cell viability is analyzed by the trypan blue exclusion method. 

Liver cells were prepared as described previously (Barth et l. 1980). Hepatocytes in suspension containing approximately 10° cells per ml L-15 medium were used for each reaction. Viability was tested by trypan blue staining. Cells above 95 % viability were used. -.. . 

Incubation of the hepatocytes, with fructose or glucose, up to 2 h, did not affect the number of hepatocytes, nor their viability, as determined by the trypan blue exclusion test. 

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