Facsymphony a5 system

In order to assess mitochondrial status, the present study tracked relative mitochondrial transmembrane potential through detecting JC-1 fluorescence. The lipophilic cation JC-1 could reversibly changes its fluorescence from green (monomeric status) to red (multimeric status) according to the variation of mitochondrial potential. JC-1 kit (Beyotime Institute of Biotechnology) was used to evaluate the mitochondrial status of luteal cells separated from the corpus luteum according to the methods provided by the manufacturer. Briefly, the separated luteal cells were digested into single cell suspension, collected and incubated with 10 μg/ml of 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylimidacarbocyanine iodide (JC-1) at 37°C, 5% CO₂ for 30 min. After washing, cells were analyzed by using a BD FACSymphony A5 system (Becton Dickinson, Franklin, NJ, USA).

For cell apoptosis evaluation, the corpus luteum was disassociated from rat ovaries on the specific day after pregnancy under dissecting microscope according to the method described by Care et al. (2013 (link)) with minor modifications. Briefly, the corpus luteum was trimmed of fat and minced into small pieces before enzymatic digestion into single cells. The minced tissues were incubated for 1 h at 37°C in DMEM F12 containing 0.1% collagenase A (Gibco), dispase (Gibco), and 25 μg/ml DNase 1 (Sigma-Aldrich). The cells were passed through a 70-μm nylon strainer (Becton Dickinson, Franklin Lakes, NJ, USA) to remove debris, and the filtrate was centrifuged at 300 g and 4°C for 5 min to pellet the suspended cells. After that, the cells were washed in FACS buffer (PBS containing 0.1% BSA). Thereafter, cells were re-suspended and stained by Annexin V-FITC Apoptosis Detection Kit (Beyotime Institute of Biotechnology, Haimen, China) according to the protocol provided by the manufacturer. The apoptotic cells were thereafter measured by using a BD FAC Symphony A5 system (Becton Dickinson, Franklin, NJ, USA).