Annexin 5 fitc pi apoptosis detection kit

CytoFLEX flow cytometer (Backman Coulter, USA) was used to detect changes of cell apoptosis. The cells from different treatment groups were collected after centrifugation (at 250xg, 4 °C, 5 min). The supernatants were discarded, and the cells were resuspend in PBS for twice to obtain cell pellet. 500µl Binding Buffer (RVBB-01, Biomiga, USA) was added to the cell pellet to resuspend the cells according to the instruction manual of AnnexinV-FITC/PI Apoptosis Detection Kit (640914, BioLegend, USA). Next, 5µl AnnexinV-FITC was added to the cells, mixed thoroughly and then further mixed with 5µl PI. The mixture was maintained at room temperature away from light for 10 min (min), and the changes of apoptosis were observed and analyzed by flow cytometry. HT-29 cells and DLD1 cells were treated by 50% inhibiting concentration (IC50) of Oxa for subsequent cell processing.

The degree of apoptosis was assessed with an Annexin V-FITC/PI apoptosis detection kit (Biolegend, San Diego, CA, USA). A total of 2 × 10⁵ cells/well were treated with crassolide for 24 h before being harvested and washed three times with PBS. The cells were incubated for ten minutes in the dark with 5 μL Annexin V-FITC (20 μg/mL) and 10 μL PI. The apoptotic cells were detected using a BD Accuri C5 cytometer, and the results were analyzed using BD Accuri C6 Software 1.0.264.21.

To detect immune cell populations in the bladder, the bladders of mice with UPEC-induced cystitis were harvested and digested with 0.1 mg/ml type D collagenase (StemCell Technologies, Vancouver, BC, Canada) for 30 min at 37°C. The digested cell suspensions were filtered through a 70-μm cell strainer. Single cells were washed twice with PBS and stained with antibodies against: CD45 (APC-R700, Biolegend), F4/80 (BV-605, Biolegend), CD11b (APC-Cy7, Biolegend), CD64 (APC, Biolegend), and Ly6G (PE, Biolegend). The macrophage and neutrophil populations were detected using a flow cytometer (BD Canto, Franklin Lakes, NJ, USA). Data were analyzed using FlowJo X software (Tree Star, Ashland, OR, USA).
Following treatment with MB49-Exo/MB49-U-Exo (10 μg/mL) or a TNFα-neutralizing antibody (Cell Signaling Technology, MA, USA) at a concentration of 100 ng/mL for 12 h, the BMDMs were washed twice with PBS and detached using trypsin-EDTA. Apoptosis was evaluated using the Annexin V (FITC)/PI apoptosis detection kit (BioLegend) according to the manufacturer’s instruction. The population of Annexin V⁺ or PI⁺ cells was detected by flow cytometry. All Annexin V⁺ cells, irrespective of their PI expression were considered to be apoptotic.

Cell death was evaluated using Annexin V‐APC/7‐AAD double staining or an Annexin V‐FITC/PI Apoptosis Detection Kit according to the manufacturer's protocol (Biolegend). Briefly, 1.0 × 10⁶ cells/ml were washed in ice‐cold PBS, centrifuged at 1000 rpm for 5 min, resuspended in APC binding buffer and incubated with AnV conjugated to APC and with 7‐AAD or PI. After 15 min of incubation in the dark, cells were diluted in 300 μl of binding buffer and analysed by flow cytometry (Becton Dickinson). For each test, 1.0 × 10⁶ cells were used, and data on at least 10,000 events were obtained using Cell Quest software (Becton Dickinson).