For cell cycle analysis, combined detached and adherent cells were fixed in 70% ethanol overnight, stained with propidium iodide (Sigma-Aldrich) and analyzed with an Accuri C6 flow cytometer. Data were collected from 20, 000 cells, and analysed with CFlow software (Becton Dickinson)36 (link).
For ɣH2AX estimation, cells were exposed to S. aureus for 2 h and were fixed in 4% paraformaldehyde/PBS followed by the permeabilization in 0.1%Triton/0.5% BSA/PBS. Cells then were incubated with Alexa Fluor 647 mouse anti-ɣH2AX (p139) antibody for 45 min and were analyzed as described above. The percent of relative phosphorylation was calculated as fold changes over the control, which was considered as 100%, and multiplied by 100. In some experiments, cells were treated with 10 mM ROS inhibitor NAC (N-acetyl-L-cysteine) (Sigma-Aldrich) 1 h before the infection. A viability and a metabolic activity of S. aureus were not affected by the treatment with NAC since there were no differences between the OD and CFUs of bacterium exposed or not to NAC.
For ɣH2AX estimation, cells were exposed to S. aureus for 2 h and were fixed in 4% paraformaldehyde/PBS followed by the permeabilization in 0.1%Triton/0.5% BSA/PBS. Cells then were incubated with Alexa Fluor 647 mouse anti-ɣH2AX (p139) antibody for 45 min and were analyzed as described above. The percent of relative phosphorylation was calculated as fold changes over the control, which was considered as 100%, and multiplied by 100. In some experiments, cells were treated with 10 mM ROS inhibitor NAC (N-acetyl-L-cysteine) (Sigma-Aldrich) 1 h before the infection. A viability and a metabolic activity of S. aureus were not affected by the treatment with NAC since there were no differences between the OD and CFUs of bacterium exposed or not to NAC.
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