Jc 1 mitochondrial assay kit

Mitochondrial membrane potential (MMP) is an important component of intermediates during OXPHOS required for ATP synthesis [39 (link)]. MMP was analysed by flow cytometry with a JC-1 mitochondrial assay kit (Beyotime Biotechnology, China) in this study. Cells were washed twice in PBS and incubated at 37°C for 30 min at the final concentration of JC-1 (1×) dissolved in the staining buffer and distilled water. The cells were washed in PBS and re-suspended in the same buffer in the culture tube for flow cytometry detection.

flow cytometry was used to analyze MMP and cell apoptosis.
MMP was examined using a JC-1 mitochondrial assay kit (Beyotime, Beijing, China) with flow cytometry (BD, United States). JC-1 forms aggregates that emit red fluorescence at high MMP while forms monomers that emit green fluorescence at low MMP (Berlepsch and Böttcher, 2013 (link)). PC12 cells were washed twice in PBS and incubated at 37°C for 30 min at the final concentration of JC-1 (1×) dissolved in the staining buffer and distilled water. The cells were washed three times in PBS and re-suspended in the same buffer in the culture tube for flow cytometry detection.
Cell apoptosis was analyzed using PE Annexin V Apoptosis detection kit (BD Pharmingen, United States), in which Annexin V was conjugated to the fluorochrome phycoerythrin (PE), to detect the phosphatidylserine (PS) on the outer membrane of the apoptosis cells (Leventis and Grinstein, 2010 (link)). PC12 cells were washed twice with cold PBS and then re-suspended in 1× binding buffer at a concentration of 1 × 10⁶ cell/mL. After that, 100 μL of cells were transferred to the culture tube and incubated with a mixture of 10 μl PE Annexin V and 7-AAD at room temperature for 20 min in the dark. Cells were subjected to flow cytometric analysis.