A modified invasion-3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay for quantitating tumor cell invasion.

Abstract

Reconstituted basement membrane matrix (Matrigel) has been utilized for in vitro assay of tumor cell invasion in recent years. In the conventional chamber for the invasion assay, however, a large number of cells passed easily through the center of the Matrigel-coated filter because the Matrigel layer could not be completely uniform by the meniscus formation. To prevent the meniscus phenomenon of the Matrigel layer, we devised a water-repellent treatment of the inside wall of the assay chamber with paraffin. Consequently, very few erythrocytes passed through the Matrigel-coated filter of this modified chamber with the erythrocyte assay, which was used to demonstrate the evenness and uniformity of the Matrigel layer on the filter. For quantitating a small number of cells which invaded through the Matrigel-coated filter by the invasion assay, a tetrazolium-based colorimetric 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) assay was used. The invasive abilities of the eight different cells were determined by this invasion-3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium+ ++ bromide assay using the modified chamber with a filter coated with 70 microliters of the 0.2-mg/ml Matrigel. After 72 h of incubation, the malignant cell lines significantly exceeded the normal cell lines in the percentage of invasion (P < 0.01). Therefore, the modified invasion-3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium+ ++ bromide assay provides a simple, easily reproducible in vitro assay for quantitating tumor cell invasion.