Trypsin edta

Dimethyl sulfoxide (DMSO), crystal violet, and trypan blue dye were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum, DMEM, RPMI-1640, HEPES buffer solution, L-glutamine, gentamycin, and 0.25% trypsin-EDTA were purchased from Lonza.

Pluronic^® F127, pluronic^® P105, MTX, triethylamine, methanol, acetone, dimethyl sulfoxide (DMSO), MTT and trypan blue were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum, DMEM, RPMI-1640, HEPES buffer solution, L-glutamine, gentamycin and 0.25% trypsin-EDTA were obtained from Lonza (Belgium). PIP (molecular weight of 285.34 Da and purity 98%) was provided by Alpha Aesar (Ward Hill, MA, USA).

All solvents and chemicals used in the present study have been obtained from Sigma Aldrich Co. Ltd. (St. Louis, MO, USA). L-glutamine, HEPES buffer solution, RPMI-1640, DMEM, gentamycin, 0.25% Trypsin-EDTA, and fetal bovine serum were purchased from Lonza.

Flow cytometry analysis (FACS) was performed for the assessment of cell viability using the FITC Annexin V Apoptosis Detection kit (BD Pharmingen, San Diego, CA, USA). Cell culture media were collected, and cells were harvested by incubation with 1 ml of 0.25% trypsin/EDTA (Lonza, Milan, Italy) for 5 min at 37°C. Trypsinization was stopped by addition of complete medium and the suspension was centrifuged at 1,200 rpm for 5 min at RT. Each pellet was washed with cold phosphate buffered saline. Then, tubes were vortexed thoroughly and centrifuged again as above. Cells were gently resuspended in 200 μl of binding buffer. Then, cell suspension was added to 5 μl of Annexin V-FITC and 10 μl of PI. Samples were mixed and incubated in the dark at 4°C for 20 min. Ten thousand cells were analyzed by FACS on a BD Accuri C6 flow cytometer to count live cells. All AnV/PI measurements were performed in duplicate, after setting the discriminator to exclude debris by forward and right angle scatter. For this assay, red fluorescence was measured corresponding to the red color of propidium iodide (FL-3 detector), and green fluorescence was measured corresponding to the green color of ANX-V (FL-1 detector). Both negative control (untreated cells) and positive control (cells treated with a high dose of H₂O₂) were included in the analysis.

Dimethyl sulfoxide (DMSO), crystal violet, and trypan blue dye were purchased from Sigma (St. Louis, MO, USA). Fetal Bovine serum, DMEM, RPMI-1640, HEPES buffer solution, L-glutamine, gentamycin, and 0.25% Trypsin-EDTA were purchased from Lonza (Basel, Switzerland). crystal violet stain (1%): It was composed of 0.5% (w/v) crystal violet and 50% methanol then made up to volume with ddH₂O and filtered through a Whatmann No.1 filter paper purchased from Sigma-Aldrich, Saint Louis, MO, USA.