Cellular glucose metabolism is essential for the reduction of cell-impermeable water-soluble tetrazolium (WST) dyes

Abstract

Water-soluble tetrazolium (WST) dyes, such as WST-1 and WST-8, are widely used in cell proliferation and anti-cell-growth drug screen assays. However, the underlying determinants for WST reduction are still largely unknown. In addition, application of tetrazolium-based assays to cellular glucose metabolism studies has not been fully explored. In the present study, we show here that WST-8 reduction is dependent on cellular glucose metabolism. In order to minimize the variance of live cell number during stimulation, we treated cells with different stimuli and performed tetrazolium-based assays within 6 hours. Withdrawal of medium glucose supply greatly attenuated WST-8 reduction but not intracellular ATP levels, while re-adding glucose reconstituted WST-8 reduction, indicating the effect was not due to cell death. The role of glucose on WST-8 reduction is specific since glutamine, fructose or galactose did not substitute for the effect of glucose on WST-8 reduction. Furthermore, inhibition of glucose transporters, intracellular glucose metabolic enzymes or EGFR-PI3K-Akt signaling also attenuated WST-8 reduction. In an attempt to screen inhibitors targeting cellular glucose metabolism from hyperglycemia-associated drugs, it turned out that HIV protease inhibitor, ritonavir, could largely block WST-8 reduction, but not cellular ATP level. Interestingly, ritonavir has been shown to acutely block glucose transport in vitro and in vivo. Taken together, our studies not only demonstrate an essential role of cellular glucose metabolism on WST-8 reduction, but also propose a novel application of tetrazolium-based assays in screening for inhibitors of cellular glucose metabolism when used in combination with ATP assay.

Novel Cell-Based Assay to Investigate Monoacylglycerol Acyltransferase 2 Inhibitory Activity Using HIEC-6 Cell Line

“…The washed cells were then treated with 50 μL of dimethyl sulfoxide (DMSO) and mixed to solubilize the formazan crystals formed by living cells. 32 Absorbance was measured at 570 nm in a microplate reader (TECAN spark cyto, Switzerland). The cell viability (%) was calculated in percentage according to the following equation …”

“…The HIEC-6 cells were seeded (5 × 10 4 cells/well) in a 96 well plate; after reaching 70–80% confluency, cells were fed with different concentrations of extracts (12.5, 25, 50, and 100 ppm) to a final volume of 100 μL and incubated for 24 h. After 24 h of incubation, the cells were rinsed with 1× PBS; then, 100 μL of MTT solution was added and incubated for 4 h at 37 °C in a CO 2 incubator (New Brunswick, Galaxy 170R, Eppendorf Company). The washed cells were then treated with 50 μL of dimethyl sulfoxide (DMSO) and mixed to solubilize the formazan crystals formed by living cells . Absorbance was measured at 570 nm in a microplate reader (TECAN spark cyto, Switzerland).…”

Guaiazulene derivative 1,2,3,4‐tetrahydroazuleno[1,2‐b] tropone reduces the production of ATP by inhibiting electron transfer complex II

“…The WST‐8 cell proliferation test we used was based on intracellular dehydrogenase activity. Generally, this method compares the number of cells with an indicated amount of dehydrogenase activity in each sample, but it also depends on the metabolic status of the cells [ 31 ]. Therefore, the number of viable cells was examined using Hoechst 33342, 48 h after the administration of 25 μ m TAT.…”

“…the metabolic status of the cells [31]. Therefore, the number of viable cells was examined using Hoechst 33342, 48 h after the administration of 25 M of TAT.…”

Mimicking of Blood Flow Results in a Distinct Functional Phenotype in Human Non-Adherent Classical Monocytes

“…Irrespective of GM-CSF activation, the metabolic activity of ex vivo cultivated monocytes was generally increased after both static and dynamic culture conditions compared to freshly isolated monocytes. According to results from tumor cell lines, an increased reduction of the tetrazolium salt WST1 was associated with increased glucose metabolism [ 19 ], indicating an increased energy demand of monocytes during suspension culture. Further studies revealed that increased glycolysis was associated with the rapid production of various metabolites that support cellular activities and cell growth [ 20 , 21 , 22 ].…”