Flow cytometric analysis of phosphorylated histone H2AX following exposure to ionizing radiation in human microvascular endothelial cells.
We applied a flow cytometric method to quantify IR-induced histone H2AX phosphorylation at serine 139 (gammaH2AX) and compared those values to those obtained using a standard microscopy based foci counting method. After PFA fixation, methanol permeabilization was suitable for both FITC- or Alexa647-gammaH2AX. In contrast, Alexa647-gammaH2AX was not suitable for ethanol permeabilization. Antibody concentrations at 1-2 microg/ml yielded the highest gammaH2AX positive percentage for both antibodies. Without DAPI staining, gammaH2AX formation can be measured as a relative fold increase. Values determined by bivariant flow cytometric analysis and those obtained using microscopic foci formation exhibited a good quantitative correlation. Values obtained by both methods could vary according to the gating or threshold setting used. gammaH2AX positive cells increased as a function of radiation dose (2-16 Gy) followed by a dose-dependent decay. The free radical scavenger N-acetyl-L-cysteine (NAC), if administered at a concentration of 4 mM 30 min before IR, was effective in reducing IR-induced gammaH2AX formation in all phases of the cell cycle. We have developed a simplified and quantitative flow cytometry based method to measure IR-induced gammaH2AX in cells and demonstrated strong correlation to values obtained by a standard automated digital microscopic foci analysis along with NIH ImageJ custom macro software.
Kataoka
Yasushi
Y
Department of Radiation and Cellular Oncology, The University of Chicago, Chicago, IL 60637, USA. ykataoka@rover.uChicago.edu
Bindokas
Vytautas P
VP
Duggan
Ryan C
RC
Murley
Jeffrey S
JS
Grdina
David J
DJ
D002478:Cells, Cultured; D004247:DNA / Q000528:radiation effects*; D053960:DNA Breaks; D004307:Dose-Response Relationship, Radiation; D042783:Endothelial Cells / Q000502:physiology* / Q000528:radiation effects*; D005434:Flow Cytometry / Q000379:methods*; D006657:Histones / Q000235:genetics* / Q000528:radiation effects* / Q000648:ultrastructure; D006801:Humans; D008833:Microcirculation / Q000166:cytology / Q000502:physiology / Q000528:radiation effects; D011829:Radiation Dosage; D011839:Radiation, Ionizing
C468783:H2AX protein, human; D006657:Histones; D004247:DNA