Bioluminescent somatic cell assay kit

ATP measurement was performed using the luciferin–luciferase reaction (Bioluminescent Somatic Cell Assay Kit, Sigma, United States). Firstly, mixed for 5 s before detection, a linear regression of standard curve containing 11 ATP concentrations from 10 fmol to 10 pmol was used to determine oocyte ATP content. Then, ATP concentrations of oocytes were measured on an EnSpire Multimode Plate Reader (PerkinElmer, United States).

ATP production was determined via bioluminescence analysis using a Bioluminescent somatic cell assay kit (Sigma-Aldrich, St. Louis, MO, Germany), according to the manufacturer’s protocol, in sorted NK cell subpopulations (HLA-DR⁺ и HLA-DR⁻) ex vivo and after 6 days stimulation with IL-2 (100 U/mL) + K562-mbIL21 cells (NK:K562 = 5:4). Then, 10⁶ cells per sample were collected, pelleted via centrifugation, added to 400 μL of lysis solution, mixed, and incubated for 4 min at room temperature. The samples were then frozen and stored at −20 °C for further analysis.
The chemiluminescence intensity was measured using a luminometer (Triathler Multilabel tester 425-004, Hidex, Finland). The ATP content in the test sample (in nmol per 10⁶ cells) was calculated in terms of proportion, taking into account the intensity of chemiluminescence and the ATP content in the control sample.

Intracellular ATP levels were determined using the Bioluminescent Somatic Cell Assay Kit (Sigma-Aldrich, USA) according to the manufacturer’s instructions.

Oocytes were individually stored at -80°C in 50 μl of ATP sample buffer (99.0 mM NaCl, 3.1 mM KCl, 0.35 mM NaH₂PO₄, 21.6 mM Na-lactate, 10.0 mM HEPES, 2.0 mM CaCl₂, 1.1 mM MgCl₂, 25.0 mM NaHCO₃, 1.0 mM Na-pyruvate, 0.1 mg/ml gentamicin, and 6.3 mg/ml BSA) [15 (link)]. A Bioluminescent Somatic Cell Assay Kit (Bioluminescent Somatic Cell Assay Kit, FL-ASC, Sigma-Aldrich, Oakville, ON, Canada) was used as per manufacturer’s instructions to assess the total ATP content per oocyte. A six-point standard curve (range 40 fmol to 125 pmol) was generated by including standards at the beginning and end in the assay. High, medium and low internal quality controls were created by processing three denuded oocytes and performing serial dilutions, as reported previously [12 (link)]. The coefficient of variation for high, medium and low internal quality controls were 6.8%, 5.6% and 3.3%, respectively.

MCF-7 cells (7 × 10⁴ cells/well) were treated by the IC50 concentration of PC for 24, 48 and 72 h, and the ATP levels (in mitochondria) measured by bioluminescent somatic cell assay kit (sigma Aldrich.MO 63103, USA).