Pb180325

After 48 h of transfection, the cells were detached with the addition of 0.25% trypsin, collected in flow tube, and centrifuged with removal of the supernatant. The cells were rinsed with PBS 3 times and centrifuged with removal of the supernatant. According to the provided instructions of the Annexin-V-FITC apoptosis detection kit (K201-100, BioVision Inc., Mountain View, CA, USA), the Annexin-V-FITC, propidium iodide (PI), and HEPES buffer were prepared at the ratio of 1 : 2 : 50 into the provided Annexin-V-FITC/PI staining solution. Next, a concentration of 1 × 10⁶ cells was resuspended in per 100 μL stainingsolution and shaken vigorously. After incubation at room temperature for 15 min, 1 mL of the HEPES buffer (PB180325, Procell Life Science & Technology Co., Ltd, Wuhan, Hubei, China) was added to the mixture for vigorous shaking. The fluorescence of FITC and PI was detected with the 525 nm and 620 nm band-pass filters at the excitation wavelength of 488 nm. The cell experiments were conducted 3 times independently.

After 48 h of transduction, cells were disrupted with 0.25% pancreatin without EDTA (YB15050057, Yubo Biotech Co., Ltd., Shanghai, China), collected in a flow tube, and centrifuged, followed by removal of the supernatant. Cells were centrifuged, and the supernatant was discarded. According to the instructions of the annexin-V–fluorescein isothiocyanate (FITC) cell apoptosis detection kit (K201-100, Biovision, Milpitas, CA, USA), the annexin-V–FITC, propidium iodide (PI), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solutions were made into annexin-V–FITC/PI dye solution at a ratio of 1:2:50. Then, 1 × 10⁶ cells were resuspended in 100 μL dye solution, shaken, and mixed with 1 mL HEPES buffer solution (PB180325, Procell, Wuhan, Hubei, China) after incubation at room temperature for 15 min. Bandpass filters of 525 and 620 nm were excited by a wavelength of 488 nm to detect FITC and PI fluorescence, respectively, for assessment of neuronal apoptosis.

Cells were digested and washed three times with cold PBS. The precipitate was resuspended with pre-chilled 70% ethanol and allowed to stand at − 20 °C overnight. According to the instructions of Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (K201-100, Biovision, Milpitas, CA, USA), the Annexin-V-FITC, propidium iodide (PI), and N-2-hydroxyethyl-piperazine-N′-2-ethane sulfonic acid (HEPES) buffer solution was used to prepare Annexin-V-FITC/PI staining solution at a ratio of 1:2:50. Each 100 μL staining solution was used for the re-suspension of 1 × 10⁶ cells, followed by incubation at room temperature for 15 min and the addition of 1 mL of HEPES buffer solution (PB180325, Procell Life Science & Technology, Wuhan, Hubei, China). The apoptosis was evaluated by detecting the fluorescence of FITC and PI at the wavelength of 525 and 620 nm, respectively. Early apoptosis was indicated by the staining of the cells with Annexin-V but not with PI (Annexin-V+/PI−), while cells that were stained with both (Annexin-V+/PI+) were considered to be necrotic or late apoptotic. Cells that were stained with PI alone or (Annexin-V−/PI+) were necrotic [36 (link)].

After transfection for 48 h, cells were detached with 0.25% ethylenediaminetetraacetic acid-free trypsin (YB15050057, Yubo Biological Technology Co., Ltd., Shanghai, China) and centrifuged with the supernatant removed. Annexin-V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) dye liquor was formulated using Annexin-V-FITC, PI and 4-(2-hydroxyerhyl)piperazine-1-erhanesulfonic acid (HEPES) at a ratio of 1: 2: 50 with reference to the manual of Annexin-V-FITC cell detection kits (K201-100, Biovision, Milpitas, CA). Each 100 µL dye liquor was adopted to re-suspend 1 × 10⁶ cells and incubated at ambient temperature for 15 min, followed by addition of 1 mL HEPES buffer solution (PB180325, Procell). Finally, cell apoptosis was assessed by detecting FITC and PI fluorescence utilizing a flow cytometer (FACS Calibur, BD Biosciences, Franklin Lakes, NJ, USA) [26 (link), 27 (link)].

After 48 h of transfection, the cells were digested with 0.25% trypsin (without ethylenediamine tetraacetic acid) (YB15050057, Shanghai Yubo Biological Technology Co., Ltd., Shanghai, China) and amassed in a flow tube. The supernatant was discarded after centrifugation. The cells were rinsed 3 times with PBS, and then the supernatant was discarded after centrifugation. According to the provided instructions of the Annexin-V-FITC Apoptosis Detection Kit (K201-100, Biovision Inc., CA, USA), the Annexin-V-FITC/PI solution was formulated with Annexin-V-FITC, PI, N-2-hydroxyethylpiperazine-N-ethane-sulphonicacid (HEPES) buffer at a ratio of 1 : 2 : 50. Every 100 μL of the dye solution was added to resuspend 1 × 10⁶ cells, followed by incubation at room temperature for 15 min. Then 1 mL of the HEPES buffer (PB180325, Procell, Wuhan, Hubei, China) was added and mixed by shaking. The FITC and PI fluorescence were examined by activating the band pass filters of 525 nm and 620 nm with a wavelength of 488 nm to examine cell apoptosis.

After transfection for 48 h, the cells were treated with 0.25% EDTA-free trypsin (YB15050057, Yubo Biotechnology, Shanghai, China). The cells were then collected in a flow tube and centrifuged, after which the supernatant was discarded. According to the protocols for the Annexin-V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (K201-100, Biovision, USA), the annexin-V-FITC, propidium iodide (PI), and 4-(2-hydroxyethyl)-1-piperazineëthanesulfonic acid (HEPES) buffer were formulated into the annexin-V-FITC/PI staining solution at a volume ratio of 1:2:50. Next, the cells were resuspended at a density of 1 × 10⁶ cells per 100 μL of the staining solution. After incubation for 15 min at room temperature, the cells were treated with 1 mL of HEPES buffer (PB180325, Procell, Wuhan, China). With an excitation wavelength at 488 nm, a flow cytometer was employed to determine FITC fluorescence by a 525 nm bandpass filter and PI fluorescence by a 620 nm bandpass filter in order to detect cell apoptosis. The experiment was repeated three times [54 (link)].