Menadione-mediated WST1 reduction assay for the determination of metabolic activity of cultured neural cells
Introduction
2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt (WST1) is a charged and water-soluble derivative of 3-(4,5-dimethylthiozal-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT). WST1 is less toxic than MTT and results in the formation of extracellular WST1 formazan without the need of cell solubilization with organic solvents [1], [2], which makes it a time-saving and non-toxic alternative to the widely used MTT reduction assay [1]. However, as the water-soluble WST1 is not taken up by cells, intracellular reduction of WST1 is prevented [3] and a membrane-permeable electron cycler is usually required to allow extracellular WST1 reduction (Fig. 1). Such electron cyclers can either be linked to plasma membrane electron transport (PMET) or permeate the cell membrane in their oxidized form [3]. In cells, oxidized electron cyclers are reduced by cellular electron sources, permeate the cell membrane in the reduced form, reduce the WST1 outside of the cells and subsequently enter the cells again to start a new cycle of electron transfer from the cells to the extracellular WST1 [4]. For the initially described WST1 assay, 1-methoxy-5-methylphenazinium methyl sulfate (M-PMS) was used as an electron cycler [1] and is still a frequently used electron cycler in commercially available WST1 reduction assay kits. Nevertheless, other electron cyclers including phenazine methosulfate (PMS) and menadione have been reported to also facilitate extracellular WST1 reduction, at least in microorganisms [5]. However, to our knowledge the use of menadione as electron cycler for WST1 reduction by mammalian cells has not been reported so far.
In order to establish a rapid and sensitive WST1 reduction assay for cultured neural cells, we have optimized the WST1 reduction assay for menadione as electron cycler using primary rat astrocytes as a cell model and have compared cell-dependent WST1 reduction in the presence of the electron cyclers M-PMS and menadione. The optimized assay was validated for its ability to detect compromised cell functions caused by exposure of astrocytes to 3-bromopyruvate (3-BP) or Triton X-100 by comparison with assays testing for cellular MTT reduction and release of cellular lactate dehydrogenase (LDH). The data presented here demonstrate that the optimized menadione-dependent WST1 reduction assay is a quick, reliable and sensitive alternative to commercially available WST1 reduction assays and other types of cytotoxicity assays.
Section snippets
Material
WST1 was obtained from Dojindo (Munich, Germany). Menadione, M-PMS, 3-BP, Triton X-100 and MTT were purchased from Sigma (Steinheim, Germany). WST8 and XTT were obtained from Cayman Chemicals (Ann Arbor, Michigan, USA). Dulbecco's modified Eagle's medium (DMEM) and penicillin/streptomycin solution were from Invitrogen-Gibco (Darmstadt, Germany). Minimal essential medium was from Life Technologies (Darmstadt, Germany). Fetal calf serum (FCS) and trypsin solution were purchased from Biochrom
Absorption spectra and calibration curves for formazan quantification
In order to record the absorption spectrum of WST1 formazan and to determine its extinction coefficient in the buffer used for cell incubations, WST1 was reduced in IB with an excess of ascorbate. The spectra of WST1 and WST1 formazan were recorded and revealed for WST1 formazan a broad absorption peak with a maximum at around 450 nm (Fig. 3). The absorbance of WST1 formazan at 450 nm increased in proportion to its concentration (data not shown). The extinction coefficient of WST1 formazan
Conclusion
We have optimized a WST1 reduction assay to establish a rapid, sensitive and reliable assay to test for intact or impaired metabolic activity of cultured neural cells within 30 min of incubation. As the use of menadione as electron cycler resulted in a doubled rate of WST1 formazan production compared to that observed with M-PMS, the replacement of M-PMS by menadione improves the sensitivity of the assay. The optimized protocol for the menadione-mediated WST1 reduction allowed detection of
Potential conflict of interest
The authors have no conflict of interest to declare.
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These authors contributed equally to this article.