Annexin 5 fitc pi double staining assay kit

Cells in early and late apoptosis were quantified using an Annexin V-FITC/PI double staining assay kit (KGA107; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) as described previously (20 (link)). Cells were harvested by trypsinization and washed three times with PBS. Subsequently, 1×10⁵ cells from each 35 mm Petri dish were resuspended in 500 µl binding buffer and stained with 5 µl Annexin V and 5 µl PI in the dark at room temperature for 20 min. The stained cells were immediately examined using flow cytometry with an excitation wavelength of 488 nm for the green fluorescence of FITC-Annexin V and 561 nm for the red fluorescence of PI.

PTX was purchased from Huafeng United Technology Co., Ltd. (Beijing, People’s Republic of China). PLGA-COOH was purchased from Shandong Institute of Medical Instruments (Jinan, People’s Republic of China). D-alpha tocopheryl polyethyleneglycol 1000 succinate (TPGS), N,N′-dicyclohexylcarbodiimide (DCC), Tween 80, N-hydroxysuccinimide (NHS), propidium iodide (PI), RNase A, Coumarin-6 (C-6), and trypsin-EDTA were obtained from Sigma-Aldrich Co. (St Louis, MO, USA). RPMI 1640 medium, penicillin–streptomycin, fetal bovine serum, and phosphate-buffered saline (PBS) were purchased from Hyclone (Logan, UT, USA). 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 4′,6-Diamidino-2-Phenylindole,Dihydrochloride were purchased from Biosharp (Hefei, People’s Republic of China). Annexin V-FITC/PI double staining assay kit was supplied by KeyGen Biotech Co. Ltd (Nanjing, People’s Republic of China). Trypan blue was purchased from TianGen (Beijing, People’s Republic of China). Taxol^® (PTX for injection) was procured from Bristol-Myers Squibb Trading Co., Ltd. (Shanghai, People’s Republic of China). All other reagents were of analytical grade.

The cell apoptosis assay was assessed using Annexin V-FITC/PI double staining assay kit supplied by KeyGEN Biotech Co. Ltd. In brief, A549 cells and A549/T cells were seeded on the 24-well plates and treated with Taxol^®, PTX-loaded PLGA NPs, and PTX-loaded PLGA-Tween 80 NPs at an equivalent PTX concentration of 1 μg mL⁻¹ for 48 hours. The control group was cells without treatment. Then, the cells were collected in 400 μL of binding buffer and mixed with 5 μL of Annexin V-FITC and 10 μL of PI for 15 minutes in the dark. The stained cells were analyzed using a flow cytometer (BD Biosciences).