Genotoxicity was evaluated by two different assays. The HCS DNA Damage Kit from Invitrogen was used to measure phosphorylation of histone variant, H2AX, which has been identified as an early response of cells to double-stranded DNA breaks. A549 cells were treated with vehicle or 100 µM etoposide for 24 hours before performing the DNA damage assay.
Figure 3A shows a quantitative representation of pH2AX-positive cells detected with secondary antibodies conjugated to either Alexa Fluor 488 dye or Alexa Fluor 647 dye and demonstrates the robustness of the assay with either secondary antibody conjugate (Z´ values of 0.9 and 0.8 for Alexa Fluor 488 and Alexa Fluor 647, respectively). A concentration dependent curve was generated and used to calculate a log EC50 value of -4.89 M (13 µM) for etoposide.
The second genotoxicity assay that was validated on the Acumen eX3 cytometer was Invitrogen’s Click- iT® EdU cell proliferation assay. This assay is superior to the standard bromodeoxyuridine assay as it negates the need for DNA denaturation, thus making the Click-iT EdU assay a more rapid and reliable option. Nascent DNA synthesis and cell proliferation were measured in cells treated with paclitaxel. The data generated demonstrated that paclitaxel could inhibit cell proliferation with a log IC50 of -8.24 M (6 nM) (Figure 3B).
This tutorial demonstrates that with its high-throughput capabilities, the Acumen eX3 laser scanning cytometer is an ideal instrument for performing in vitro, cell-based, high-content drug toxicity profiling. Each of the cytotoxicity assays was performed using protocols that analyzed every cell contained in each well, using instrumentation that was able to capture, normalize, and export data quickly.
All of the Invitrogen assays validated on the Acumen eX3 cytometer were shown to be robust with excellent screenability. Finally each of the reagents was suitable for both fixed and permeabilized cells, which enables efficient multiplex interrogation of off- and on-target effects of compounds for toxicological profiling.