Luciferase based enhanced atp assay kit

JC-1 mitochondrial membrane potential measurement kits (Beyotime, China) were used to measure mitochondrial membrane potential (ΔΨm). Briefly, TM3 cells (2 × 10⁵) were resuspended in 0.5 mL JC-1 working solution and incubated at 37 °C for 20 min. The cells were washed with buffer twice and then analyzed by flow cytometry (BD Biosciences, USA). A luciferase-based enhanced ATP assay kit (Beyotime, China) was used to determine ATP levels. Briefly, the cells were washed with cold PBS in 6-well plates and lysed immediately in 200 μL lysis buffer on ice. The lysate was centrifuged at 12,000 g for 5 min. 20 μL of the supernatant was added into a 96-well plate containing 100 μL ATP detection working solution. The luminescence was detected by a multi-function microplate reader (Perkinelmer, USA). The protein concentration of each group and an ATP standard curve were used to calibrate the ATP levels in the cells. The cellular ATP levels were presented as the percentage of the level observed in the control group.

A JC-1 MMP measurement kit (Beyotime, Shanghai, China) was used to measure MMP (ΔΨm). Briefly, the indicated cells (2×10⁵) were resuspended in 0.5 mL of JC-1 working solution and incubated for 20 min. The cells were washed and analyzed by flow cytometry (Biosciences, Franklin Lakes, NJ).
A luciferase-based enhanced ATP assay kit (Beyotime, Shanghai, China) was used to determine ATP levels. Briefly, the indicated cells were lysed and centrifuged at 12,000× g for 5 min. The supernatant was added to a 96-well plate containing ATP detection working solution. Luminescence was detected by a multifunction microplate reader (Perkin Elmer, USA). The protein concentration of each group and an ATP standard curve were used to calibrate the ATP levels in the cells.

The ATP levels in spermatozoa were determined using a luciferase-based enhanced ATP assay kit (Beyotime, China) and BCA assay kit (Beyotime, China). Briefly, after incubation with EV and EV electroporated with circ-CREBBP siRNA, spermatozoa were washed with PBS and lysed immediately in 200 µL lysis buffer. Then, the lysate was collected and centrifuged at 12,000×g for 5 min at 4 °C. Then, 20 µL of supernatant was added to the well containing 100 µL ATP detection working dilution. The luminescence was detected by a multifunctional microplate reader (Biotek, USA). The protein concentration of each group was measured, which was used to calibrate the ATP levels in cells.