New Soluble-Formazan Assay for HIV-1 Cytopathic Effects: Application to High-Flux Screening of Synthetic and Natural Products for AIDS-Antiviral Activity

Abstract

We have developed an effective and optimally safe microculture method for rapid and convenient assay of the in vitro cytopathic effects of human immunodeficiency virus (HIV-1) on human lymphoblastoid or other suitable host cells. The assay procedure is applicable to the evaluation of drug effects on in vitro infections induced directly in cultured host cells by cell-free HIV-1 or by coculture with H9 cells chronically infected with HIV-1. The assay uses a newly developed tetrazolium reagent that is metabolically reduced by viable cells to yield a soluble, colored formazan product measurable by conventional colorimetric techniques. This simple microassay minimizes the number of plate manipulations typically required with other assay methods and, coupled with computerized data collection and analysis, facilitates large-scale screening of agents for potential antiviral activity. To support and enhance the discovery of new anti-HIV-1 agents, the National Cancer Institute is offering investigators worldwide the opportunity to submit new candidate agents for anti-HIV-1 screening with this method.

Potent and selective activity of a new carbocyclic nucleoside analog (Carbovir: NSC 614846) against human immunodeficiency virus In vitro

“…Data are derived from the MTA as previously described (18). An XTT-PMS solution is prepared immediately prior to its addition to the wells of the culture dish [i mg/ml XTT (19,20) solution in media without FCS].…”

Cytotoxicity, Apoptosis Induction and Anti-Metastatic Potential of Oroxylum indicum in Human Breast Cancer Cells

“…Extracts were tested for its cytotoxicity by XTTformazan dye formation assay (Weislow et al, 1989). MDA-MB-231 and WRL-68 cells were seeded in their respective culture medium (200 µl, 1 x 10 4 cells/well and 6 x 10 3 cells/well respectively) in a 96-well plate and incubated at 37 ºC for 24 h with/without 5% CO 2 supply.…”

“…Cytotoxicity was estimated by measuring the amount of resultant XTT-formazan as a consequence of XTT reduction by succinate dehydrogenase in living cells (Weislow et al, 1989). All four tested extracts showed a concentration-dependent increase in cytotoxicity with increasing doses in both the cell lines, MDA-MB-231 and WRL-68 (Figure 1).…”

Hydrosoluble formazan XTT: its application to natural products drug discovery for Leishmania

“…To avoid this problem, MTT analogs such as sodium-2,3-bis-[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and more recently MTS, have been effectively employed in cell proliferation assays. The reduced form of these agents is a watersoluble formazan, which obviates the need for solubilization (Weislow et al, 1989). An additional advantage of the XTT and MTS methods is the shorter period of incubation which results from the addition of the electron coupling agent PMS to accelerate the reduction of the substrate (Buttke et al, 1999).…”

“…In this experiment, 250 parasites per well were seeded in two 96-well plates and cultured for 6 days. Three replicates were processed with XTT substrate every 24 h, as described previously (Weislow et al, 1989). Briefly, 50 Al of PBS containing 1.48 mM XTT activated with 200 AM phenazine methosulfate (PMS, Sigma) was added daily.…”