Neutral red (NR) assay for cell viability and xenobiotic-induced cytotoxicity in primary cultures of human and rat hepatocytes

Abstract

Neutral red (NR) in medium was absorbed and concentrated in lysosomes of cultured rat and human hepatocytes. NR uptake increased with the time of incubation and reached a plateau in 2 hr. Uptake was proportional to the concentration of the NR solution and the numbers of viable liver cells. Prolonged culture of hepatocytes increased the numbers of lysosomes, and thus, the dye accumulation. The NR can be extracted from lysosomes for quantitative measurement of hepatocyte viability and cytotoxicity of xenobiotics. With this assay, several serum-free media (e.g., Waymouth's, MEM, LHC-8, etc.) were compared for the maintenance of viable hepatocytes in vitro. Interestingly, LHC-8 medium, which is used to grow human bronchial epithelial cells, best preserved viable rat hepatocytes. The cytotoxic effects of dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) were examined by NR assay on rat and human hepatocyte cultures and were found to be dependent on dose and time of the exposures. NR50 was 20 mM for DMN and 0.072 microM for AFB1 in rat hepatocytes with 24 hr of exposures and reduced to 12.5 mM for DMN and 0.053 mu microM for AFB1 with 48 hr exposures. Human hepatocytes were more resistant to the toxicity of both chemicals; NR50 values were 100 mM DMN and 1.8 microM AFB1 respectively, for 24 hr treatments. Compared with lactate dehydrogenase (LDH) leakage test, the NR assay was simpler and more sensitive in determining the viability and cytotoxicity of xenobiotics in primary cultures of hepatocytes.

Modulation of COX-2 Expression by Statins in Human Aortic Smooth Muscle Cells

“…The concentration of ethanol or Me 2 SO did not exceed 0.3% and did not alter COX expression. The absence of cellular toxicity by statins, isoprenyltransferase inhibitors, and toxins was evaluated by neutral red assay (15).…”

Growth substrate induced functional changes elucidated by FTIR and Raman spectroscopy in in–vitro cultured human keratinocytes

“…AB was added to the HaCaT cells in DMEM F-12 (15.6 g/L; Sigma-Aldrich) which did not contain phenol red (as this interferes with the fluorescence of resorufin) and NaHCO 3 (1.2 g/L) giving a pH of 6.9 ± 0.2. Neutral Red (NR; 3-Amino-7-dimethylamino-2-methyl-phenazine hydrochloride) is a supravital fluorescent dye which is weakly cationic and can diffuse across the cellular membrane, accumulating in cellular lysosomes [31][32][33]. Increases in cell wall permeability and lysosome fragility are associated with the latter stages of cell death, thus the level of accumulation of NR in the cell is indicative of the level of viability in a sample of cells.…”

“…Increases in cell wall permeability and lysosome fragility are associated with the latter stages of cell death, thus the level of accumulation of NR in the cell is indicative of the level of viability in a sample of cells. Neutral Red stock solution was prepared using 0.5 mg of NR dye in 100 ml DMEM-F12 minus phenol red indicator [31][32][33].…”

“…Samples were then washed once in 2.5 ml PBS and 2.5 ml of a fixative solution containing 1% glacial acetic acid and 50% ethanol in dH 2 O, to lyse the cell and release the NR dye. Cells were then shaken at 240 rpm for 30 mins and fluorescence was read at 650 nm after excitation at 540 nm [31][32][33]. Commassie Blue is a dye which binds to protein within the lysed cell [34].…”

Micrococcal nuclease (endonuclease) digestion causes apoptosis and mitotic catastrophe with interphase chromosome condensation in human Chang liver cells

“…1C). NR uptake is an established test of cell viability, not cell death (Goldberg and Frazier, 1989;Zhang et al, 1990). NR enters the cell via endocytic internalization that is dependent on active membrane flow and fusion mechanisms in viable cells.…”