An optimized method for measurement of gamma-H2AX in blood mononuclear and cultured cells

Abstract

Phosphorylation of histone protein H2AX on serine 139 (gamma-H2AX) occurs at sites flanking DNA double-stranded breaks (DSBs) and can provide a measure of the number of DSBs within a cell. We describe a flow cytometry-based method optimized to measure gamma-H2AX in nonfixed mononuclear blood cells as well as in cultured cells, which is more sensitive and involves less steps compared with protocols involving fixed cells. This method can be used to monitor induction of gamma-H2AX in mononuclear cells from cancer patients undergoing radiotherapy and for detection of gamma-H2AX throughout the cell cycle in cultured cells. The method is based on the fact that H2AX like other histone proteins are retained in the nucleus when cells are lysed at physiological salt concentrations. Cells are therefore added without fixation to a solution containing detergent to lyse the cells along with a fluorescein isothiocyanate-labeled monoclonal gamma-H2AX antibody, DNA staining dye and blocking agents. The stained nuclei can be analyzed by flow cytometry to monitor the level of gamma-H2AX to determine the level of DSBs and DNA content and to determine the cell cycle stage. The omission of fixation simplifies staining and enhances the sensitivity. This protocol can be completed within 4-6 h.

FOXO3 Transcription Factor Is Essential for Protecting Hematopoietic Stem and Progenitor Cells from Oxidative DNA Damage

“…␥H2AX nuclear foci were analyzed by immunofluorescence staining using a rabbit polyclonal antiphospho H2AX (Ser-139) (Millipore) and imaged on a Leica DMRA2 fluorescence microscope using ϫ400 oil immersion objective. Freshly isolated bone marrow cells stained for HSPC were fixed in 2% paraformaldehyde and stained overnight at 4°C with 1:100 mouse monoclonal anti-H2AX pS139 FITC conjugate (Millipore) (or anti-H2AX pS139 APC conjugate, Biolegend) in BLOCK9 solution as described previously (54). The samples were next diluted into PBS, 2% FBS and analyzed by FACS.…”

Novel HSP90 inhibitors, NVP-AUY922 and NVP-BEP800, radiosensitise tumour cells through cell-cycle impairment, increased DNA damage and repair protraction

“…The cells were then incubated in the same medium under standard conditions and analysed by flow cytometry 30 min, 1 and 2 days after IR exposure. For analysis, cells were trypsinised, washed twice in PBS, fixed and stained for gH2AX, according to a protocol described elsewhere (Muslimovic et al, 2008). The cells were then counterstained with propidium iodide (Sigma P-4170, 10 mg ml À1 ) in the presence of ribonuclease A (Sigma R-5250, 25 mg ml À1 ) as described elsewhere (Djuzenova et al, 1994 …”

Low-dose DNA damage and replication stress responses quantified by optimized automated single-cell image analysis

“…The advantages of our new method compared with the alternative, currently available approaches based on flow cytometry, physical methods such as electrophoresis, and expensive complex scanning devices 9,18,19,30,35 include: (1) high sensitivity; (2) quantitative data output; (3) low investments for setting up the method (based on standard fluorescence microscope equipped with digital camera and common PC with software available in most laboratories); (4) automation of the routine that allows analysis of thousands of cells per day; and (5) relatively simple operation and high degree of transportability among laboratories, thanks also to our detailed protocols (see the Suppl. information) with both written guides and illustrative movies to facilitate the widespread use of the technique.…”

“…[9][10][11] Techniques to assess the DDR markers are evolving, and particularly for nonadherent cells such as lymphocytes, recently optimized protocols based on flow cytometry are convenient, albeit not particularly sensitive means for measurements of g-H2AX. 35 On the other hand, analysis of DNA damage at very low doses, and assessment of the vast majority of cell types that are naturally adherent (for which detachment from substratum is very stressful) require alternative methods which are however presently limited to very costly and sophisticated scanning instruments that require large investments and specially trained personnel. To overcome these limitations, we have developed and report here a versatile, highly sensitive method to quantify various parameters of the DDR in large numbers of adherent individual cells with statistical robustness, at very low cost affordable to most, if not all laboratories, with high degree of automation, and easily usable by the many researchers who are familiar with common fluorescence microscopes and personal computers.…”