Abstract
Alamar Blue, an oxidation-reduction indicator, added to a culture medium is reduced in living cells as an intermediate electron acceptor in the mitochondrial electron transport system, so that the absorbance of alamar Blue at a specific wavelength changes with the progression of cell growth or the number of living cells. In order to rapidly and quantitatively evaluate the development of preimplantation mouse embryos cultured in vitro, a colorimetric assay with alamar Blue was applied. Two-cell embryos, obtained from CD-1 mice, were cultured in 4-well dishes with 1 ml of Whitten's medium containing 3 mg/ml of bovine serum albumin, and then transferred to 96-well plates (5, 10 or 20 embryos/well) with 100 μl of Whitten's medium containing alamar Blue (10%, v/v) and 3 mg/ml of bovine serum albumin. First, the effect of alamar Blue on the development of mouse embryos was examined. Although alamar Blue strongly inhibited the subsequent preimplantation development of the late 2-cell embryos, it had no significant effect on that of the 4- to 8-cell embryos. Changes in the absorbance of alamar Blue, with the progression of embryonic development and the number of embryos per well, were then monitored at 24-h intervals by measuring the absorbance using a microtiter plate reader. When the development of the 4- to 8-cell embryos was evaluated by the alamar Blue assay, the method required only 5-20 embryos per well of a 96-well plate and allowed daily monitoring of the proliferation for a period of 72 h, without compromising the sterility of the cultures. The absorbance of alamar Blue changed with the progression of embryonic development and correlated with the number of embryos per well. The stimulatory effect of EDTA on the development of zygotes obtained from CD-1 (5 or 10 embryos/well) was also determined by this method. The results indicate that the assay is useful for quantifying the development of mouse embryos cultured in vitro.
