Cell counting kit 8 cck 8

Cetyltrimethyl ammonium bromide (CTAB), tetraethoxysilane (TEOS), and Hoechst 33342 were purchased from Sigma-Aldrich (St Louis, United States). Indocyanine Green (ICG) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD) were purchased from Absin (Shanghai, China). Human cervical cancer cells (Hela) were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The membrane protein extraction kit, calcein-AM/PI Double Staining Kit, Cell Counting Kit-8 (CCK-8) and penicillin–streptomycin were purchased from Biyuntian (Jiangsu, China). Dulbecco’s modified Eagle medium (DMEM) was purchased from Gibco (Grand Island, United States). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, United States). All other reagents were of analytical grade without any purification.

A549 and NCI-H1299 cell proliferation was assessed by Cell Counting Kit-8 (CCK-8, Biyuntian, Beijing, China). Briefly, tumor cells were seeded in 96-well plates (2000 cells/well) and cultured with RMPI 1640 complete culture medium supplemented with 10% FBS. Cell proliferation was examined at 0, 24, 48, and 72 hours according to the manufacturer’s protocol and absorbance was quantified at 450 nm by microplate reader (Biorad, NJ, USA). For the colony formation, A549 and NCI-H1299 cells were seeded in 6-well plates at 500 cells/well and cultured in RMPI complete medium in a humidified incubator. After 10 days, colonies were fixed with paraformaldehyde and stained with crystal violet (Biyuntian, Beijing, China). Visible colonies were counted. Each experiment was performed three times independently.

Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8, Biyuntian, China). Briefly, 2 × 10⁴ SHEDs were inoculated on samples in 48-well plates. The samples were then cultured for 1, 4, and 7 days, respectively, and incubated in CCK-8 working solution for 2 h. The culture medium was replaced every 2 days. Then, 100 μL of the sample solution was transferred to a 96-well plate and the absorbance was measured at 450 nm. The relative growth rate (RGR) was calculated using the following equation: RGR (%) = OD_test/OD_control × 100%.
In morphological studies, cells were treated for 15–30 min with a Calcein-AM fluorescent dye kit (HR0444, Baiaolaibo, China) after 7 days of cell culture. The Calcein-AM staining solution was removed and the cells were washed twice with a serum-free medium. Following the preparation of Hoechst working solution (C1017, Biyuntian, China) at a concentration of 20 μg/mL, the cells were further incubated for 10–20 min. The cells were then washed twice with PBS before being examined under inverted fluorescence microscopy (IX73, Olympus, Japan). RGR was calculated using OD_control represents the absorption of the sample at 12 h.

The cell proliferation rate was determined using the Cell Counting Kit-8 (CCK-8; Cat#C0038, Biyuntian Biotechnology Co., Ltd., Shanghai, China) in accordance with the manufacturer’s protocol. Briefly, SCLC cells were seeded into the wells of 96-well plates at a density of 5000 cells per well. Cells were cultured in the TA-SCs medium (SC/DMS114-con med, SC/DMS53-con med, and SC/H1048-con med; 10% v/v), the SCs medium (SC-con med, 10% v/v), and the RPMI-1640 medium supplemented with 2% FCS (medium, 10% v/v). After 24 h, 10 μL of CCK-8 solution was added to all wells and the cells were incubated for 2 h. The resulting color was evaluated at a wavelength of 450 nm using iMark™ Microplate Absorbance Reader (Bio-Rad Laboratories, Hercules, CA, USA). Each assay was performed in triplicates and repeated two times.

SA (200–500 Pa·s) was purchased from Shanghai Taitan Technology Co., Ltd., Shanghai, China, anhydrous calcium chloride (CaCl₂) was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd., Shanghai, China and Polosham 188 (F-188) was purchased from Shaanxi Zhengyi Pharmaceutical Accessories Co., Ltd. Carbon support copper mesh (230 mesh) and phosphotungstic acid were obtained from Beijing Zhongjing Keyi Technology Co., Ltd., Beijing, China. 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil, cell membrane green fluorescent probe), Hoechst 33342, 4% paraformaldehyde fix solution, antifade mounting medium and Cell Counting Kit-8 (CCK-8), were all purchased from Biyuntian Biotechnology Co., Ltd., (sShanghai, China) 5(6)-aminofluorescein was bought from Nanjing Xinfan Biotechnology Co., Ltd., Nanjing, China. Polycarbonate film was bought from Whatman Company, City, UK. Dialysis bag (Cut-off molecular weight = 3500Da) was obtained from United States for carbonization. Fetal bovine serum (FBS), RPMI 1640 medium, and DMEM medium were ordered from the Shanghai Chenyi Biotechnology Company, Shanghai, China. Trypsin and penicillin-streptomycin were purchased Yingjie Jieji (Shanghai) Trading Co., Ltd., Shanghai, China.