Cflow software

The flow cytometry analysis of STLV-1 Env expression was performed by an established method as described previously [25 (link)]. In brief, cells were pretreated with FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany). For the staining of HTLV-1 Env, mouse monoclonal anti-HTLV-1 gp46 Ab [67/5.5.13.1] (Abcam, Cambridge, United Kingdom) [25 (link)], mouse IgG1 kappa monoclonal Ab (isotype) (Abcam, Cambridge, United Kingdom), and goat anti-mouse IgG-PE Ab (Abcam, Cambridge, United Kingdom) were used in accordance with the manufacturer’s protocol. After staining, a flow cytometric analysis was immediately performed on a BD Accuri C6 flow cytometer with CFlow software (Becton Dickinson, Franklin Lakes, NJ, USA), and the collected data were analyzed by FCS Express 4 (De Novo Software, Los Angeles, CA, USA).

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.

The change in cell size was determined by flow cytometric measurement of changes in the forward light scatter (FSC) axis of the neutrophils (Accuri C6 flow cytometer; Becton, Dickinson and Company, San Diego, CA, USA). Mean FSC values for stimulated neutrophils were compared with mean FSC values of unstimulated cells in the medium control. Overlapping histograms were generated using the C flow software (Version 1.0.264.21, Becton, Dickinson and Company, San Diego, CA, USA) to make a graphical comparison.

Differences between two groups were assessed for statistical significance by Student’s t-test for each cell line independently. TMZ dose response data and sphere growth data were analyzed using a one-way analysis of variance (ANOVA) with post-hoc Tukey test using MATLAB software (MathWorks, Natick, MA). Neurosphere apoptosis data were analyzed using one-way ANOVA with post-hoc Fisher’s least significant difference test. Flow cytometry data was analyzed using either FlowJo software (Tree Star Inc, Ashland, OR) or CFlow software (Becton Dickinson Biosciences, Franklin Lakes, NJ). Differences were considered statistically significant at p < 0.05. Data shown represents the mean ± standard deviation of three independent experimental replicates.

Following 15d-PGJ₂ or MG132 treatments (18 h) and activation with TNF- α (0.2 ng/ml for 6 h), the EC were harvested to be stained for flow cytometric analysis. To block non-specific binding, EC were incubated in 2% BSA in PBS (blocking buffer) for 15 min before adding the antibodies. FITC-VCAM-1, PE-ICAM-1, and APC-E-selectin antibodies (BD, Rankling Lakes, NJ, USA) were used to label EC in 2% BSA in PBS. The antibodies were incubated for 30 min at room temperature. Following two washes with blocking buffer, the cells were fixed in 2% paraformaldehyde. Forward and side scatter gates were established to exclude non-viable cells and cell debris from the analysis. The mean fluorescence intensity of 2 × 10⁵ cells was analyzed in each sample. Auto-fluorescence signals generated by unlabeled cells were used as negative controls in each experiment. Flow cytometric analysis was performed on an Accuri C6 instrument and analyzed with CFlow^® Software (Accuri, Ann Arbor, MI, USA). The data were expressed as median fluorescence intensity of three independent experiments.