Propidium iodide rnase

A2780-EV and A2780-COMMD1 cells were harvested after 24 hours of incubation with 2 μM cisplatin and fixed in ice-cold 70% ethanol. Cells were stained with mouse anti-phospho-Ser10-Histone H3 antibody and with rabbit anti-phospho-Ser139-H2AX and subsequently stained with Alexa-488-conjugated anti-mouse and Alexa-647-conjugated anti-rabbit antibody. In addition, counterstaining was performed with propidium iodide/RNAse (Sigma-Aldrich). Cell cycle distribution, phospho-Histone H3 and anti-phospho-H2AX positivity were analyzed on a FACSCalibur (Becton Dickinson Biosciences) equipped with CellQuest software. Per sample, at least 10,000 events were analyzed, and indicated results show averages and standard deviations of three independent experiments.

Percentages of apoptotic cells and cell cycle phases were determined using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) after the cells were stained with Annexin-V FITC Apoptosis Kit (Invitrogen, Carlsbad, CA) and propidium iodide/RNase (Sigma-Aldrich, St. Louis, MO), respectively, as we described recently [56 (link)]. All experiments were carried out in triplicate with separate cultures and all data were analyzed with Flowjo (Ashland, OR).

Cells were fixed in ice-cold 70% ethanol, and incubated overnight at 4 °C. Subsequently, cells were stained for rabbit anti-phospho-histone H3 antibody (1:100, Cell Signalling, #9701) and mouse MPM2 (1:100, Millipore, 05-368). After washing with PBS–0.05% Tween-20, cells were incubated with Alexa 488-conjugated and Alexa 647-conjugated secondary antibodies (1:100, Molecular Probes) and counterstained with propidium iodide/RNase (Sigma). FACS analyses were performed on a FACS-Calibur (Becton Dickinson) and samples were analyzed using Cell Quest software. Data were analyzed using FlowJo software. At least 10,000 events were analyzed per sample.