Accutase

The functional activity of P-gp was evaluated using the previously described method [49 ]. The HBL-100/Dox cells were detached with accutase (HiMedia Laboratories, Maharashtra, India). For efflux assay, the cells were first loaded for 20 min with a cold culture medium containing 5.0 μg/mL of Rhodamine 123 (Rh123; Sigma–Aldrich, St. Louis, MO, USA). After incubation, the cells were washed twice and divided into several fractions (2.5 × 10⁵ cells per point). One fraction was incubated in a pure medium, and the other one was incubated with MK (2.0 μM) added. The well-known P-gp inhibitor verapamil (30.0 μM; Alfa Aesar, Ward Hill, MA, USA) served as a reference control. Incubation was carried out in culture medium RPMI-1640 without FBS at 37 °C for 60 min. Cell fluorescence was evaluated on CytoFlex S flow cytometer (Beckman Coulter, Brea, CA, USA). The results were analyzed using FlowJo software (ver. X 10.0.7r2, FlowJo Software, San Jose, CA, USA) and were represented as geometric mean fluorescence intensity (gMFI) and MAF coefficient [50 (link)], which were calculated using the following formula: MAF = (Mean (inh.) − Mean (free))/Mean (inh.), where MAF is the MDR activity functional, mean (free) is the mean value of cell fluorescence without inhibitor, and mean (inh.) is the mean value for cell fluorescence with inhibitor.

The Human Glioblastoma Cell line LN-18 was obtained from American Type Culture Collection (ATCC Rockville, USA). The Human Glioblastoma tumor tissue samples were collected from surgeries performed at Sasoon hospital, DY Patil Hospital and Inamdar hospital, Pune. Informed consent was obtained from patients for tissue procurement in accordance with the protocol approved by the institutional ethics committee of NCCS and graded by pathologist. Primary Cultures were obtained by processing GBM tumor samples using Accutase (Himedia) and Zymefree (Himedia) to obtain adherent cell cultures which were passaged to 3–10 passages. We have previously reported the expression of neuronal markers in primary cultures-G162 (link). Cells were maintained in Dulbecco’s modified eagle’s medium (DMEM) with 4 mM L-glutamine, 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, supplemented with 5% heat inactivated fetal calf serum (Gibco) in a humidified incubator at 37 °C with 5% CO₂. Cells were dislodged using trypsin (0.125%) – EDTA (0.02%) solution.

Briefly, the HBL-100 and HBL-100/Dox cells (1 × 10⁶ cells per well) were seeded into a 6-well plate (SPL Life Sciences, Pocheon-si, Republic of Korea). After 24 h, the cells were treated with MK (at IC₅₀ and 2 × IC₅₀ concentrations) for another 24 h period. The cells were detached with accutase (HiMedia Laboratories, Maharashtra, India) and then fixed with 70% ice cold ethanol followed by overnight incubation at 4 °C. The cells were washed twice with PBS, permeabilized using 0.1% Triton X-100 in PBS solution, and subsequently stained with 5 μL of the BD Horizon™ BV605 Rabbit Anti-Active Caspase-3 antibody (BD Biosciences, Franklin Lakes, NJ, USA). The cells were then washed, resuspended in PBS, and analyzed using a CytoFlex S flow cytometer (Beckman Coulter, Brea, CA, USA). Cell fluorescence was evaluated on a CytoFlex S flow cytometer (Beckman Coulter, Brea, CA, USA). The results were analyzed using FlowJo software (ver. X 10.0.7r2, FlowJo Software, San Jose, CA, USA) and represented as geometric mean fluorescence intensity (gMFI).