Annexin 5 fitc pi apoptosis detection kit

The cytotoxicity of empty NPs against A549 cell and A549/taxol cell was evaluated using WST-1 assay.33 Briefly, A549 cell and A549/taxol cell were seeded onto 96-well plates at a density of 5.0×10⁴ cells per well and cultured with 100 μL of DMEM medium at 37°C in 5% CO₂ atmosphere for 24 h in overnight cultures. Then, the original medium was removed and the cells were incubated with fresh medium containing empty nanoparticles at different concentration, respectively. After 12 h and 24 h of incubation, 10 μL of WST-1 solution was added to each well and cultured for additional 4 h. Afterward, the cell viability was determined by measuring the absorbance at 450 nm using a micro-plate reader (Model 680; Bio-Rad, Hercules, CA, USA). The relative cell viability (%) was calculated using the following equation (3):
$\text{The cell viability}=\frac{{\mathrm{OD}}_{\text{treat}}-{\mathrm{OD}}_{\text{blank}}}{{\mathrm{OD}}_{\text{control}}-{\mathrm{OD}}_{\text{blank}}}\times 100$
For apoptosis analysis, A549/taxol cell (1×10⁶) was seeded on 6-well plates and cultured with various concentrations of CS-ALG@TPGS-PLGA NPs at 37°C. After 48 h of incubation, the cells were harvested by trypsin digestion and centrifugation. Then the cells were washed three times with PBS. Finally, Annexin V-FITC/PI apoptosis detection kit (Becton-Dickinson, NJ, USA) was used to analyze the apoptotic efficacy by a flow cytometer (FACSCalibur).