Apoptosis detection kit

ELE was purchased from Dalian Huali Jingang Pharmaceutical Co. Ltd (Dalian, China). CTX was purchased from Hubei Qianmo biological technology Co. Ltd (Hubei, China). Soya lecithin (the main ingredient is phosphatidylcholine, PC) was purchased from Shanghai Taiwei pharmaceutical Co. Ltd (Shanghai, China). d-α-tocopherol polyethylene glycol succinate (TPGS) was purchased from Wuhan Guobangda pharmaceutical chemical Co. Ltd (Wuhan, China). Medium chain triglyceride (MCT) was purchased from Xinxing Tieling pharmaceutical Co. Ltd (Tieling, China). Cholesterol (Chol) was purchased from Beijing Dingguo Changsheng Biotechnology Co. Ltd (Beijing, China). DSPE-PEG2000-transferrin was purchased from Xi’an Ruixi Biological Technology Co. Ltd (Xi’an, China). DAPI and GAPDH were purchased from Sigma-Aldrich (St Louis, USA). Penicillin–streptomycin and high-glucose DMEM, trypsin EDTA and FBS were purchased from Gibco Life Technologies (AG, USA). CCK-8 was purchased from Meilunbio (Dalian, China). Cypate was purchased from Hangzhou Xinqiao Biotechnology Co. LTD (Hangzhou, China). Coomassie blue, RIPA lysis buffer and bicinchoninic acid protein kit (BCA) were purchased from Biosharp (Shanghai, China). Apoptosis detection kit was purchased from Genview (USA).

Cytotoxicity effects of ELE/CTX@LIP, ELE/CTX@BLIP, Tf-ELE/CTX@LIP and Tf-ELE/CTX@BLIP on RG2 cells were determined using a CCK-8 kit. RG2 cells (3000 cells/well) were inoculated in 96-well plates and incubated for 24 h. After incubation, cells were exposed to ELE/CTX@LIP, ELE/CTX@BLIP, Tf-ELE/CTX@LIP and Tf-ELE/CTX@BLIP (CTX concentrations ranging from 0.4 to 200 ng/mL) for 48 h. Non-treated cells were used as negative controls. Medium was evaluated as the blank control. Cytotoxicity was quantitatively determined by measuring the absorbance at 450 nm using a Spark multi-functional microporous plate testing platform (Tecan, Switzerland).
Furthermore, apoptotic cells were determined by FACS analysis (CytoFLEX S, USA). RG2 cells were incubated with ELE/CTX@LIP, ELE/CTX@BLIP, Tf-ELE/CTX@LIP and Tf-ELE/CTX@BLIP and 50 ng/mL CTX for 48 h then treated with the apoptosis detection kit (Genview, USA) for 10 min. Percentage of apoptotic cells was determined using a FACS Calibur System. Non-treated cells were used as the negative control [46 (link)].