Cells were seeded at 0.8-1 × 104/ml per well in 96-well tissue culture plates. Post 24 h, 10% FBS-containing medium was replaced with 2% FBS-supplemented media cells and different concentrations of PGG or PI extract (0.1, 0.3, 1, 3, 10, 30, and 100 μg/ml) for the indicated time. Hydroxychloroquine sulfate (CQ) at 10 μM and wortmannin (WTM) at 500 nM were treated before 1 h of PI treatment in cells. Three hours before the indicated time period, Alamar blue at a working concentration of 15 μg/ml was added to the respective media. The fluorescence units (FU) were recorded in an EnVision multi-plate reader (PerkinElmer, Waltham, MA, United States) at excitation and emission wavelengths of 560 and 590 nm, respectively. Percent cell viability is calculated using the following formula:
% Cell viability = [(FU Treated- Blank)/(FU Untreated- Blank)] × 100.
The half-maximal inhibitory concentration was determined in GraphPad Prism 8.0 by plotting a non-linear dose–response curve.
For apoptosis evaluation, cells were stained with FITC Annexin V-Propidium iodide (PI) according to the manufacturer’s protocol (Invitrogen). Cells were acquired in a Attune acoustic focusing flow cytometer (Thermo Fisher, United States) and analyzed in FCS Express software version 7 (De Novo Software, Los Angeles, CA).
% Cell viability = [(FU Treated- Blank)/(FU Untreated- Blank)] × 100.
The half-maximal inhibitory concentration was determined in GraphPad Prism 8.0 by plotting a non-linear dose–response curve.
For apoptosis evaluation, cells were stained with FITC Annexin V-Propidium iodide (PI) according to the manufacturer’s protocol (Invitrogen). Cells were acquired in a Attune acoustic focusing flow cytometer (Thermo Fisher, United States) and analyzed in FCS Express software version 7 (De Novo Software, Los Angeles, CA).