Atp bioluminescent somatic cell assay kit flasc

The luciferin/luciferase detection of ATP was performed with the microplate reader Infinite F200 Pro (TECAN, Lyon, France) with ATP bioluminescent somatic cell assay kit (FLASC, Sigma, Saint-Quentin Fallavier, France). Breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-435S transfected with siRNA control (siC) or siRNA targeting IP₃R3 (siR3) were seeded at 2000 cells/well in white 96-well Nunc dishes with clear bottoms 72 hours before ATP measurements. For extracellular ATP measurements, 100μl of supernatant were incubated with the luciferin/luciferase (FLAAM, Sigma) at a final concentration of 0.04%. For intracellular ATP measurements cells were incubated with 100 μl of ATP releasing reagent (FLSAR, Sigma), before incubation with the luciferin/luciferase (FLAAM, Sigma). To determine the amount of ATP released, a calibration curve was constructed using known concentrations of ATP in solution (1, 10, 100, 1000, 10 000, 100 000 pM). Control experiments were performed to eliminate any drug effect on luciferase activity.

Incubation was performed for 90 min under capacitation or no-capacitation conditions, with and without spermine at 1.0 mM, spermidine at 0.1 mM, and putrescine at 1.0 mM. Immediately thereafter, the sperm suspensions (25 μL) were transferred to a conical tube, and the extraction buffer (0.1 M Tris-HCl, 4 mM EDTA, pH 7.8) was added, followed by boiling at 100 °C for 5 min. Subsequently, the sperm suspensions were centrifuged at 20,000× g for 5 min, and the supernatant was recovered. Using a luciferase bioluminescence assay, the ATP levels were measured in accordance with the manufacturer´s protocol (ATP Bioluminescent Somatic Cell Assay Kit FL-ASC; Sigma Chemical Co.). Luminescence was measured using a Synergy 2 Multi-Function Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA) [55 (link)].