In vivo, according to the instructions, renal tissue cell apoptosis was investigated by terminal deoxynucleotidyl transferase‐mediated dUTP nick-end labeling (TUNEL) staining using the DeadEnd™ Fluorometric TUNEL System (Promega, Wisconsin, USA). Detection of positive cells was implemented with by fluorescence microscopy (Carl Zeiss, Jena, Germany) at ×200 magnification. For each sample, 10 images were randomly selected for counting to identify the amount of apoptotic cell nuclei. In vitro, TUNEL staining was carried out using the Fluorescein (FITC) TUNEL Cell Apoptosis Detection Kit (Servicebio, Wuhan, China). The ratio of TUNEL‐positive cells was evaluated in 3 visual fields.
The apoptosis rate of cultured TCMK-1 cells was assessed by an Annexin V-FITC/propidium iodide (PI) kit (4A Biotech, Beijing, China) in combination with flow cytometry (Beckman Coulter, Brea, CA). The preparation of cells was performed following the protocol of manufacturer. In brief, cells were washed twice with PBS and then resuspended in 1×Binding Buffer (100 μl). Afterwards, the cell suspension was added with Annexin V (5 μl) and incubated at room temperature in darkness for 15 min. Lastly, PI (5 μl) and 1× Binding Buffer (400 μl) were inserted and the cells were analyzed by flow cytometry within 1 h.
The apoptosis rate of cultured TCMK-1 cells was assessed by an Annexin V-FITC/propidium iodide (PI) kit (4A Biotech, Beijing, China) in combination with flow cytometry (Beckman Coulter, Brea, CA). The preparation of cells was performed following the protocol of manufacturer. In brief, cells were washed twice with PBS and then resuspended in 1×Binding Buffer (100 μl). Afterwards, the cell suspension was added with Annexin V (5 μl) and incubated at room temperature in darkness for 15 min. Lastly, PI (5 μl) and 1× Binding Buffer (400 μl) were inserted and the cells were analyzed by flow cytometry within 1 h.