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.
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.