Pbs buffer

PLGA (50:50 7 kDa–17 kDa, acid terminated), polyethylenimine (25 kDa, branched), epirubicin and paclitaxel were purchased from Sigma Aldrich (Darmstadt, Germany). Acetone was purchased from Sisco Research Laboratories Pvt. Ltd. (Mumbai, India). PF68 surfactant, Alamar blue, PBS buffer and sodium bicarbonate were procured from HiMedia (Mumbai, India). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) were obtained from Gibco Life Technologies (Carlsbad, CA, USA). In-house distilled water was used during the studies.

0.5 × 10⁶ cells were seeded in 60 mm cell culture dish and cultured overnight for the attachment. Next, cells were washed with cell culture grade 1X phosphate buffered saline (PBS) buffer (TS1101, Himedia, India) before the treatment to remove residual FBS. Respective cells treatment was performed in serum free DMEM high glucose containing 4 mM l-glutamine, 100 IU/ml penicillin/100 μg/ml streptomycin. Untreated cells were consider as untreated control in experiments. Cell viability assay was performed using propidium iodide (PI) (P4170; Sigma-Aldrich, St. Louis, Missouri, USA), flow cytometric analysis. Samples from different groups were collected by trypsinization, and washed twice with cold PBS buffer. Cells were re-suspended in 100 µl PBS and added PI with final concentration of 1 µg/ml and incubate at 2–8 °C for 5 min in dark. Cell analysis was performed on BD FACS Calibur flow cytometer (BD Biosciences). All experiments were performed in triplicate.