Doxorubicin-treated H9c2 cells: caution with luminescent ATP and Hoechst 33258 assays

摘要

High throughput, plate-based cellular assays are becoming increasingly popular due to rapidity of data generation and the opportunity for a higher number of replicates than possible with older methods. Assays for DNA content based on Hoechst 33258 (H33258) fluorescence (Rago et al. 1990) and ATP content using a kit based on a series of coupled reactions producing chemiluminescence in response to ATP levels (such as Cell Titer-Glo, Promega, Madison, WI) are popular biomarkers used in high throughput assays. We found when using doxorubicin as a toxicant in initial experiments that H33258 fluorescence was extremely low, while ATP content based on luminescence was higher than expected. To investigate the DNA results, eight concentrations of DNA ranging from 0.024 to 3.0 μg/0.1 ml dissolved in water were incubated with doxorubicin at 1 or 5 μM for 4 h prior to addition of the H33258. Fluorescence at 458 nM (with excitation at 358 nM) was measured as per the published protocol (Rago et al. 1990). Water or 1 or 5 μM doxorubicin-only blanks were subtracted from the raw data. Fluorescence results are shown in Fig. 1. If a point in the figure lacks SEM bars, they were too small to appear outside of the data point. At all DNA concentrations, fluorescence was significantly lower (p<0.0002, Mann–Whitney test) with added doxorubicin at 1 or 5 μM compared to fluorescence without doxorubicin, except at the lowest DNA concentration in the 5-μM doxorubicin group. To investigate apparent enhanced ATP content with doxorubicin treatment, H9c2 cardiomyoblasts (ATCC) seeded in 96-well plates were treated with 1 or 5 μM doxorubicin for 24 h. Results of ATP determination (Cell Titer-Glo, Pomega) were found to be higher when doxorubicin was left in the well vs. replacement with serum-free media just prior to the ATP assay (Fig. 2).