Annexin 5 pi staining kit

Kidney tissue was dissected into small pieces and gently minced by gentleMACS Dissociator (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) in Hank’s buffer containing 10% type IV collagenase (Gibco). Red blood cells were lysed in both kidney and blood samples. Single cells were divided into groups and stained by fluorescence-conjugated antibodies including CD45-APCCy7 (BD Pharmingen, San Diego, CA, USA), CD11b-APC (BD), FPR2-AF647 (Bioss, Beijing, China), CD86-BB700 (Novus), CD163-AF405 (BD), and CD62L-BV421 (BD) for 30 min in staining buffer (BioLegend, San Diego, CA, USA). Neutrophil Elastase-PE (Bioss), MPO-FITC (BioLegend), and CD68-PE (Invitrogen) were stained after fixation/permeabilization using fixation and fixation/permeabilization buffer (BioLegend) according to the manufacturer’s protocols. Apoptosis assay was conducted using commercially available Annexin V/PI staining kit (Beyotime) according to the manufacturer’s instructions. Experimental data were acquired using FACS Aria III (BD Biosciences, San Jose, CA, USA) and analyzed by Flow Jo X (BD Biosciences).

TCMK-1 cells were cultured in six-well plates and harvested using trypsin without EDTA before and after staining. Cellular ROS, mitochondrial ROS, apoptosis, and MMP were determined using a Reactive Oxygen Species Assay kit (Beyotime Institute of Biotechnology, Shanghai, China), MitoSOX kit (Life Technologies, Carlsbad, CA, USA), Annexin V-PI staining kit, and JC-1 detection kit ((Beyotime Institute of Biotechnology, Shanghai, China), respectively. The cells were then stained according to the respective manufacturer’s instructions and analyzed via flow cytometry. Ten thousand events were recorded using the FACSCalibur system (BD Biosciences, CA, USA), and the resulting data were analyzed using FlowJo software.

To characterize the expression of surface markers, the MSCs were immune-stained and detected by flow cytometry analysis. Antibodies used in this detection are anti-CD73-PE, anti-CD90-FITC, anti-CD105-PE, anti-CD14-FITC, anti-CD34-FITC, anti-CD19-PE, anti-CD45-APC and anti-HLA-DR-PE (Biolegend, San Diego, CA, USA).
To assess the apoptotic rates, H9c2 cells were seeded in a 6-well plate at 30,000 cells/well for 24 h and then desired treatments were performed for 24 h. Cells were stained using an Annexin V/PI staining kit (Beyotime, Shanghai, China) following the manufacturer’s instructions and detected by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).