Hydrogen peroxyde

Tetrachloroauric acid trihydrate (99.99%), sodium citrate dihydrate (≥ 99%), Triton X-100 (laboratory grade), sodium borohydride (≥ 98%), silver nitrate (> 99%), poly(ethylene glycol) methyl ether thiol (mw 2000), poly(allylamine) hydrochloride (PAH, mw 50000), sodium poly(4-styrene sulfonate) (PSS, mw 70000), nitric acid (≥ 65%), nitric acid (1M), hydrochloric acid (≥ 37%), sulfuric acid (95.0–97.0%), hydrogen peroxyde (30% w/w), ethanol (≥ 99.8%), 2-propanol (99.5%), and ethyl cellulose (10 cP; 22 cP; 46 cP; 100 cP) were bought from Sigma Aldrich (Milano, Italy); α-mercapto,ω-carboxy poly(ethylene glycol) (mw 3000), poly(ethylene glycol) methyl ether thiol (mw 5000), poly(ethylene glycol) methyl ether thiol (mw 10000), and poly(ethylene glycol) methyl ether thiol (mw 20000) were bought RAPP Polymere (Tübingen, Germany); Ethylene glycol (99.5%) and ammonia solution in water (30% w/w) were bought from Carlo Erba Reagenti S.p.A. (Milano, Italy) Glass coverslides (22 × 26 mm, 0.14 mm thickness) were bought from Delchimica Scientific Gòassware (Napoli, Italy).

Generation of reactive oxygen species (ROS) was measured by increase of fluorescence of the cell-permeable superoxide sensitive probe dihydroethidium (DHE; Sigma). CML cells were stained with 2 μM DHE for 15 min at 37 °C in culture medium without FCS. After washing, 5.10⁵ cells were cultured for 24 h in complete culture medium and treated with CDKs inhibitors for various times or concentrations, as indicated, in a final volume of 1 mL. Then, cells were washed twice with sterile PBS 1X, resuspended in 0.5 mL PBS and analyzed for fluorescence distribution using a Beckman-Coulter XL4 flow cytometer. Positive control of ROS generation was made treating cells with 5 μL of hydrogen peroxyde (Sigma).