A rapid and sensitive fluorometric screening assay using YO-PRO-1
to quantify tumour cell invasion through Matrigel
Clin Exp Metastasis, 14 (5):
451-8 (October 1996)Gohla, A Eckert, K Maurer, H R Research Support, Non-U.S. Gov't England
Clinical & experimental metastasis Clin Exp Metastasis. 1996 Oct;14(5):451-8..Abstract
A new quantitative assay for the study of tumour cell invasion in
vitro is described. Employing the novel fluorescent dye YO-PRO-1,
cells that penetrate Matrigel-coated transwells are counted on the
basis of dye-bound cellular nucleic acid content. Following transmigration,
the cells in the lower compartments are lysed by freezing in water.
After a brief incubation with YO-PRO-1, nucleic acid or DNA content
is measured as fluorescence intensity in 96-well microplates and
quantitated by a cell- or DNA-calibration curve. Using standard curves,
a linear relationship between fluorescence intensity and cell number
was found in the range tested (from 100 to 80 000 cells). The mean
relative intra- and inter-assay variability of the cell quantitation
in this range was 3.5 and 4.2%, respectively. When applied to Matrigel
invasion studies, as few as 400 cells could be counted. The quantitation
could be performed within 3 h. HCT 116, MDA MB 231 and HT 29 cells
were investigated as examples of tumour cells with different invasive
abilities in the 48-h Matrigel invasion assay. Using YO-PRO-1, 6.5
+/- 0.6% invasive HCT 116 cells and 52.6 +/- 4.5% MDA MB 231 cells
(percentage of the inoculated cell population) were measured. HT
29 cells were practically non-invasive. These results were confirmed
by visual scoring of DAPI-stained nuclei. In conclusion, the main
advantages of the assay are its sensitive, reproducible and rapid
quantitation of tumour cell invasion in vitro and the applicability
to extended sample numbers by measuring in 96-well microplates.