Annexin 5 fitc

Cells transfected with plasmids were digested with 0.25% trypsin (Gibco) and washed twice with PBS. Next, cells were added to 100 μL of binding buffer to prepare a suspension at a density of 1 × 10⁶ cells/mL, and the suspension was inoculated with Annexin V-FITC (Yeasen Biotech Co., Ltd.) and PI at room temperature in the dark for 5 min. Finally, cell apoptosis was measured using the FC500MCL flow cytometry system. The assay was repeated three times to obtain the mean value.

Flow cytometry was conducted as described²³ (link). The total cells treated with or without XS561 were collected with centrifugation at 400 × g.
For apoptosis detection, cells were stained with Annexin V/FITC for 15 min, followed by staining of propidium iodide (PI) for 5 min (Yeasen Biotechnology). After fluorescence compensation, cells were instantly detected through FITC and PC5.5 fluorescence channels. The proportion of early apoptosis (PI^–/Annexin V⁺) and late apoptosis (PI⁺/Annexin V⁺) was determined as the percentage of apoptotic cells.
For mitochondrial membrane potential (ΔΨm) assay, cells were stained with 5 μg/mL 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1, ThermoFisher Scientific) for 30 min, and subsequently detected by FITC and PE fluorescence channels.
For mitochondrial reactive oxygen species (Mito-ROS) detection, cells were stained with Mito-SOX (Thermo Fisher Scientific) for 20 min, followed by PE fluorescence channel detection. Data were analyzed using a cytoFLEX Flow Cytometry System (Beckman–Coulter, Miami, FL, USA).

Flow cytometry was conducted as described²³ (link). The total cells treated with or without XS561 were collected with centrifugation at 400 × g.
For apoptosis detection, cells were stained with Annexin V/FITC for 15 min, followed by staining of propidium iodide (PI) for 5 min (Yeasen Biotechnology). After fluorescence compensation, cells were instantly detected through FITC and PC5.5 fluorescence channels. The proportion of early apoptosis (PI^–/Annexin V⁺) and late apoptosis (PI⁺/Annexin V⁺) was determined as the percentage of apoptotic cells.
For mitochondrial membrane potential (ΔΨm) assay, cells were stained with 5 μg/mL 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1, ThermoFisher Scientific) for 30 min, and subsequently detected by FITC and PE fluorescence channels.
For mitochondrial reactive oxygen species (Mito-ROS) detection, cells were stained with Mito-SOX (Thermo Fisher Scientific) for 20 min, followed by PE fluorescence channel detection. Data were analyzed using a cytoFLEX Flow Cytometry System (Beckman–Coulter, Miami, FL, USA).

Cell apoptosis was detected using Annexin V-FITC/PI Apoptosis Detection Kit (YEASEN, Shanghai, China). After a routine incubation in a 6-well culture plate for 48 h, adherent and suspended cells were harvested, washed twice with Phosphate Buffered Saline (PBS) and then resuspended with Binding Buffer. Subsequently, suspended cells were incubated for 10 min with Annexin V-FITC and propidium iodide (PI, Yeasen Biotechnology Co., Ltd., Shanghai, China) in a dark room for 15 min. Next, cell apoptosis assay was carried out by using FACScan flow cytometry (Becton Dickinson, Immunocytometry System, San Jose, CA).

The transfected cells were digested with 0.25% trypsin, washed twice with PBS, added using 100 µl binding buffer and prepared into 1×10⁶ cells/ml suspension. Annexin V-FITC and PI (product no. 40302ES20; Shanghai Yeasen Biotechnology Co., Ltd.) used according to the manufacturer's instructions, were sequentially added, and incubated 5 min at room temperature under dark conditions. Detection was performed using a FC500MCL flow cytometer and CytExpert software (version 2.0; Beckman Coulter, Inc.). FlowJo version 10.0 (Tree Star, Inc.) was also used for analysis. The experiment was repeated 3 times and data were averaged.