Phospho-H2AX Activation Kit

The kit contains a primary monoclonal antibody that detects only the phosphorylated form of human H2AX, a goat anti-mouse secondary antibody conjugated to DyLight™ 549 Fluorophore and various other reagents and buffers required for immunofluorescence staining for high-content screening (HCS) assays.

The nucleosome is made of four core histone proteins (H2A, H2B, H3 and H4). H2AX belongs to a H2A family of histones.  DNA damage induction by various agents leads to rapid phosphorylation of H2AX at Ser139 (also known as Gamma H2AX) by ATM, ATR or DNA protein kinase leading to the formation of DNA foci at the site of DNA double-strand breaks (DSBs).  Phosphorylated H2AX helps in recruiting the proteins responsible for double-strand break repair.

Phosho-H2AX in the nucleus of A549 cells treated with etoposide was assayed using the Phospho-H2AX Activation Kit, the Cellomics ArrayScan® HCS Reader and the Compartmental Analysis BioApplication Software Module. Induction of DNA damage by etoposide leads to phosphorylation of H2AX. The output parameter for this assay is the nuclear intensity of phosphor-H2AX staining after treatment. Cells labeled using this kit also can be imaged by fluorescence or confocal microscopy.

 
 
Figure 2. Staining of phospho-H2AX in A549 cells treated with vehicle (control; 0.1% DMSO in media)(left) or with 50 µM etoposide for 1 hour (right). Cells were stained according to the kit protocol and imaged using a Cellomics ArrayScan HCS Reader. Dose response curve for etoposide-treated A549 cells (bottom). Data represents mean ± SD from three plates (eight wells per 96-well plate per dose of etoposide). EC₅₀ = 68.21 ± 1.25 µM.

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