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A Modified Invasion-3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazolium Bromide Assay for Quantitating Tumor Cell Invasion¹

The First Department of Surgery, Kagoshima University School of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890, Japan

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 µI 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.

¹ This study was supported in part by a Grant-in-Aid for Scientific Research and for Cancer Research from the Ministry of Education, Science and Culture of Japan.

² Present address: Department of Surgery, The Johns Hopkins University School of Medicine, Ross Building Room 754, 720 Rutland Avenue, Baltimore, Maryland 21205. To whom requests for reprints should be addressed, at First Department of Surgery, Kagoshima University School of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890, Japan.

Received 2/ 8/94. Accepted 5/ 4/94.

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