Annexin 5 fitc pi apoptosis detection kit

Poly(amidoamine) (PAMAM) dendrimer with a ethylenediamine core (generation 5 with 128 surface amino groups) was purchased from Sigma (Shanghai, China). Nimotuzumab (h-R3) was a gift from BioTech Pharmaceuticals Co., Ltd. (Beijing, China). The reporter plasmid, pEGFP-N1, was purchased from Invitrogen (Carlsbad, CA, USA). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and dimethylsulfoxide (DMSO) were from Sigma (Shanghai, China). The fluorescent dyes (Cy5 and Hoechst 33342) were purchase from Fanbo Biochemical Co. (Beijing, China). Annexin V-FITC/PI Apoptosis Detection Kit was got from Solarbio LIFE SCIENCES (Beijing, China). The plasmid pFLAG-CMV2-p53 encoding p53 was a gift from Prof. Sun of Tsinghua University, recombined from pFLAG-CMV2 and wild-type human p53 cDNA. Paclitaxel injections were purchased from Haikou Pharmaceutical Factory Co., Ltd (Haikou, China). All other reagents were obtained from the Biodee Reagent Company (Beijing, China).

The apoptosis of tumour cells (A549) treated by different concentrations of compound 5k, was measured with Annexin V–FITC/PI apoptosis detection kit (Solarbio, Beijing, China), according to instructions of kit, and detected by BD Accuri C6 flow cytometry (American BD Corporation Shanghai Co., Ltd.)

Cell apoptosis was analyzed with an Annexin V‐FITC/PI Apoptosis Detection Kit (CA1020) produced by Solarbio (China). After 48 hours of transfection in each group, cells were made into a cell suspension with a density of 1 × 10⁵ cells/ml using 1× Binding Buffer. Then, 5 μl of FITC Annexin V and 5 μl of propidium iodide (PI) were added to 100 μl of cell suspension, and the cells were subsequently incubated in the dark for 15 minutes. Finally, a CytoFLEX Flow cytometer (Beckman Coulter, USA) was used to detect the apoptosis of cells in each group and calculate the apoptosis rate.

According to the instruction of annexin V-FITC/PI Apoptosis Detection Kit (Solarbio), cells were tripsinized and washed twice with PBS. The cells were then resuspended in 200 μL binding buffer with 10 μL annexin V-FITC and 10 μL PI, gently mixed and incubated for 15 min in the dark at room temperature. Binding buffer (300 μL) was added to each tube and the samples were analyzed with flow cytometry Becton Dickinson FACStarPLUS (Becton Dickinson) within 1 h.

Two human glioma (SW1088 and HS683) cell lines were inoculated into 6-well plates, and then the cells were cultured in a CO₂ incubator at 37 ^∘C. According to different experimental groups, cells were subjected to knockdown and overexpression treatment. Then, the subsequent experiments were carried out according to the instructions of annexin V-FITC/PI apoptosis detection kit (Solarbio, CA1020). First, collect the cell culture solution into a new tube, wash the cells with PBS, and then add EDTA-free trypsin to digest the cells. Subsequently, the collected cell culture medium was added to terminate the digestion, and the cells were collected into the centrifuge tube. After centrifugation at 1000 g for 5 minutes, the supernatant was discarded and the cells were collected. The cells were resuspended with PBS and counted. Resuspended cells (5× 10⁴) was centrifuged again at 1000 g for 5 minutes, the supernatant was discarded, and 195 µL annexin V-FITC binding solution was used to resuspend the cells. Then add 5 µL of annexin V-FITC and mixed well. Finally, add 10 µL of propidium iodide (PI) staining solution and mixed well. After incubation at room temperature for 15 minutes in the dark, apoptosis was detected by flow cytometry.