Image pro plus software program

In each well of a 6-well plate, cells (8×10³) were suspended in Basal Medium Eagle (BME) medium (1 mL, with 10% FBS and 0.33% agar) and plated over a layer of solidified BME (3 mL, with 10% FBS and 0.5% agar). The cultures were incubated in a 37°C, 5% CO₂ incubator for 7 d and colonies in soft agar were counted under a microscope equipped with the Image-Pro Plus software program (Media Cybernetics, Bethesda, MD).

To assess cell apoptosis, dechorionated live embryos at 24 hpf were incubated in 5 μg/mL acridine orange (AO, CAS #10127–02-3, Sigma-Aldrich) solution in the dark at room temperature for 1 h. The embryos were then washed with EM three times for 5 min each, anesthetized with 0.02% MS-222 for 5 min, and mounted laterally in 1% w/v low melt agarose on a microscope slide for imaging under a fluorescent microscope. Further, AO positive cells in the eye region were quantified using Image Pro Plus software program (Media Cybernetics, Inc., Silver Spring, MD). The activity of caspase-3, an important marker of apoptosis, was also measured in larvae at 120 hpf based on a previously published method (Wang et al., 2013 (link)). In brief, the activity of caspase-3 was determined by a colorimetric assay based on the ability of caspase-3 to hydrolyze the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) and release a yellow formazan product (pNA), which had a high absorbance at 405 nm. A 5-point calibration curve of pNA was included in each assay and caspase-3 activity was expressed as nmol/mg protein. Tissue samples from 20 larvae per treatment group were pooled as one replicate, and the test was repeated three times each with larvae from different batches.