Facscanto flow cytometer

The intracellular production of ROI was measured by flow cytometry using oxidation-sensitive fluorescent probe hydroethidine (HE, Sigma). After 7 days, MDM expanded alone or in the presence of TMVs (added in the described regimen) were preincubated with 10 µM HE for 30 min and stimulated with phorbol 12-myristate 13-acetate (4 µM PMA, Sigma) for 10 min, after which cells were washed and analysed immediately with FACSCanto flow cytometer.

Asyncronized DAOY and ONS-76 cells were treated with 4-HPR (5–10 μM) or Vincristine (50 μM) as positive control, for 24h trypsinized, and fixed in cold 70% ethanol. DNA was stained with 100 μg/ml propidium iodide (PI) (Sigma- Aldrich) in hypotonic citrate buffer with 20 μg/ml ribonuclease A. Stained nuclei were analyzed for DNA-PI fluorescence using a FACSCanto flow cytometer. Resulting DNA distributions G0/G1, S, G2/M and apoptotic phase of the cell cycle were analyzed with FACSDiva software. 4-HPR effects on cell cycle were also investigated by detection of phosphorylated ciclin-kinase2 (anti-phospho-Chk2, Cell Signaling Technology, Danvers, MA) levels by cytofluorimetric analysis. Cells treated with 10 μM Etoposide were used as positive control. Briefly, cells were detached, collected by centrifugation and fixed/permeabilized using Cytofix-Cytoperm solution (BD) for 10 minutes at 4°C at dark. Unconjungated phospho-Chk2 primary antibody was added to each tube for 1h at room temperature. Cells were then washed and incubated with the secondary antibody conjugated with Alexa Fluor 482 (Life Technologies, Monza, Italy) for 30 minutes at room temperature. Cells were then rinsed, resuspended in PBS 1X and analyzed by flow cytometry. Expression levels of Cyclin D1 and CDK4 were detected by Western Blot analysis (see below).

Intracellular staining was performed on cells fixed in ice cold 70% ethanol and blocked in perm/wash buffer (BD Biosciences, 554722) containing 10% human AB serum for 1 h. Cells were labeled for 1 h with appropriate primary antibodies, or isotype control antibody, at room temperature in the dark. Cells were subsequently washed and resuspended in 100 μL PBS for analysis by flow cytometry. To assess viability cells were labeled with annexin V (Becton Dickinson, Franklin Lakes, NJ, USA) and propidium iodide (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer's instructions and analyzed using a FACSCanto flow cytometer.