Comparison of Three Fluorescence Microplate Assays for Assessment of HD Cytotoxicity

The cytotoxic effect of sulfur mustard (HD) was examined in cultured human epidermal keratinocytes (HEK, Clonetics #2971, of neonatal origin). Three different cytotoxicity assays of adherent cells in 96-well culture plates were compared, i.e., procedures employing alamar blue, calcein-AM or ethidium homodimer (EthD-1) as the fluorescent probe. The overall data indicate that HD cytotoxicity in adherent HEK can be assessed using either alamar blue or calcein-AM. Alamar blue is more sensitive than calcein-AM and is therefore recommended for general use. Alamar blue also offers the additional advantage that it is non-toxic and freely diffuses from cells in both oxidized and reduced forms. It can therefore be used to obtain replicate analyses on the same cell preparations (following washout from the medium). The assessment of HD cytotoxicity using adherent HEK in multiwell plates is rapid, does not require sophisticated instrumentation, and is free of cellular damage produced by detachment procedures required for flow cytometry analyses. Difficulties associated with the multi-well plate technique for the assay of HD cytotoxicity are relatively minor and involve the use of suboptimal controls.