Cck 8 cell viability assay

CCK-8 cell viability assay was performed, as described by the manufacturer (Dojindo Laboratories, Kumamoto, Japan). NSCLC cells were seeded into 96-well plates (1x10⁴ cells/well) and treated with various concentrations of crizotinib or DMSO for 24 h in the presence or absence of nivolumab. Similarly, the flow cytometry-based cytotoxicity was performed, as described protocol. Briefly, the target cells were labeled with CFSE (1 x 10⁶ cells in 1 ml PBS with 0.5 uM CFSE, 20 min, 37˚C in the dark) and washed twice with warm culture medium. CFSE-labeled 5 x 10⁴ tumor cells were incubated at various concentrations of crizotinib for 24 h with CIK cells pre-incubated with 20 μg/mL nivolumab or IgG 4 isotype (24 h) to perform redirected cytolysis assay at an E/T ratio of 10:1. Following 24 h of culturing, the cells were stained with Hoechst 33258 (Cayman Chemical, Hamburg, Germany) and were quantified using BD FACS Canto II. The absolute number of 3000 beads (Biolegend, San Diego, CA) was acquired by a BD Canto II cytometer. Then the absolute numbers of cells per uL were analyzed by FlowJo V10 software (Tree Star, Ashland, Oregon). The absolute number of cells were calculated according to Precision Count protocol provided by Biolegend Company as follows:

NP cytotoxicity in the rat hepatocyte (BRL-3A) cell line was determined using a cell counting kit-8 (CCK-8) cell viability assay (Dojindo Molecular Technologies, Kumamoto, Japan) [47 (link)]. NP suspensions were sterilized under ultraviolet light for 30 min before experiments. The BRL-3A cells were first seeded into 96-well plates at a density of 5.5 × 10³ cells per well and incubated at 37°C in a 5% CO₂ atmosphere for 24 h. Wells without cells acted as blank controls. Different concentrations of PLGA-PFOB NPs and cRGD-PLGA-PFOB NPs in a solution medium (20, 10, 5, 2.5, 1.25 mg/mL) were prepared to replace the previous medium. After 24 h, 10 μL CCK8 reagent was added to each well and incubated for 4 h at 37°C. The absorbance of each well was measured at 450 nm with a microplate reader (EL×800 Universal Microplate Reader, BIO-TEK Instrument Inc, Winooski, VT, USA). All experiments were performed in triplicate.