H5110-08Y1: Histone H2A.X, phosphorylated (S139) Mab (FITC) 1x370ul Supplied as a liquid in PBS, 5mg/ml BSA, 0.05% sodium azide. Store at -20°C.

H5110-08Y2: IgG, Normal mouse (FITC) 1x350ul of FITC-conjugated mouse antibody Supplied as a liquid in PBS, 5mg/ml BSA, 0.05% sodium azide. Store at -20°C.

H5110-08Y3: Fixation Solution, 16X. 1x5ml Supplied as a liquid in 37% formaldehyde, 10-15% methanol. Store at room temperature.

H5110-08Y4: Wash Solution, 10X. 1x2ml Supplied as a liquid in PBS, 1% saponin. Store at room temperature.

H5110-08Y5: Supplied as a liquid: 100mM HEPES, pH 7.4, 1.4M sodium chloride, 25mM CaCl2, 5% saponin, 0.2um filter sterilized. Store at room temperature.

Aliquot and store H5110-08Y1 and H5110-08Y2 at -20°C. Store other components at RT. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.

Kit Description Quantity: Sufficient reagents for 100 assays per kit. Storage: Upon receipt, refer to individual components for storage conditions. Anti-phospho Histone H2A.X, FITC conjugate and Normal Mouse IgG, FITC conjugate may be stored at 4°C for up one month, however storage at -20°C is recommended. Aliquot FITC-conjugated antibodies to avoid repeated freeze-thaw cycles. All other components are stored at room temperature.

Use: The H2A.X Phosphorylation Assay Kit (Flow cytometry) is a cell based assay formatted for flow cytometric detection of levels of phosphorylated Histone H2A.X. Cells are cultured in microplates, treated with agents that induce DNA damage or apoptosis, which stimulates H2A.X phosphorylation. Cells are then fixed and permeabilized in preparation for staining and detection. Histone H2A.X phosphorylated at serine 139 is detected by the addition of the anti-phospho-Histone H2A.X, FITC conjugate. Cells are then scanned in a flow cytometer to quantitate the number of cells staining positive for phosphorylated Histone H2A.X. Please refer to the User Manual for further information and a detailed assay procedure.

Technical Information for Kit Components Anti-phospho-Histone H2A.X, FITC conjugate (mouse monoclonal IgG1) Product Description: Mouse monoclonal IgG1 specific for human histone H2A.X phosphorylated at serine residue 139, conjugated to fluorescein isothiocyanate (FITC). Immunogen: KLH-conjugated, synthetic peptide (C-KATQA[pS]QEY) corresponding to amino acids 134-142 of human histone H2A.X. Broad species cross-reactivity is expected based on conservation of amino acid sequence.

Normal Mouse IgG, FITC Conjugate Mouse IgG purified by ion-exchange chromatography from pooled mouse serum, conjugated to fluorescein isothiocyanate (FITC).

Quality Control Testing Log phase Jurkat cells were treated with staurosporine (1ug/ml) for the indicated time. Histone H2A.X (Ser139) phosphorylation was detected as described in the assay manual and the cells were analyzed on a Becton-Dickinson. See picture 1.