The P5 mouse temporal bone was micro-dissected in ice-cold HBSS (Thermo Fisher Scientific). The inner ear was opened from its apex to base. After removal of the bone, the exposed BM and SV were dissected separately and put into an incubation chamber. For testing ATP release, the isolated BM and SV was incubated in a zero Ca2+ solution (ZCS) containing (in mM): 137 NaCl, 5.36 KCl, 0.44 KH2PO4, 0.18 Na2HPO4, 0.1 EGTA, 25 HEPES, and 5.55 Dextrose (pH 7.3). To quantify ATP release, the BM and SV were incubated in ZCS for 20 min at 37°C, 5% CO2. The collected incubation solutions were kept on ice. The amount of ATP was measured by a bioluminescence method with a luciferin-luciferase assay kit (FL-ASC, Sigma, USA) using a black 96-well plate to avoid optical cross-talk. The bioluminescence was read by a Biotek Synergy 4 Hybrid Microplate Reader (Biotek Instruments Inc, Winooski, VT, USA). All bioluminescence measurements reported in this article fell within the linearity range of the ATP standard curve generated according to the manufacturer’s instructions.
Fl asc
Manufactured by Merck Group
Sourced in United States
The FL-ASC is a laboratory instrument designed for fluorescence-based analyses. It is a versatile tool that can be used for a variety of applications, including cell-based assays, protein analysis, and nucleic acid detection. The FL-ASC provides accurate and reliable measurements, enabling researchers to obtain high-quality data for their scientific investigations.
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5 protocols using fl asc
1
Quantifying ATP Release from Cochlear Tissues
2
Measuring Oocyte ATP Levels
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MII-stage oocytes were washed with PBS-PVA collected for ATP measurement, ATP levels were determined using a commercially-available adenosine 5′-triphosphate (ATP) bioluminescent somatic cell assay kit (FLASC, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Briefly, oocytes were transferred into a 96-well plate with 45.8 μL ATP assay buffer, then 0.2 μL ATP probe, 2 μL ATP converter, and 2 μL developer mix were added. The plate was placed at room temperature for 30 min. ATP levels were measured using a luminometer (Bioluminat Junior, Berthold, Germany).
3
Quantifying ATP Levels in Oocytes and Cumulus
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ATP levels in oocytes and cumulus cells were measured using a kit (FL-ASC from Sigma Aldrich) as previously described with a minor change [65 (link)]. Briefly, 30 denuded oocytes or cumulus cells collected from 30 cumulus-oocyte complexes in each mouse were snap-frozen in a microfuge tube containing 50 μL water and stored at −80°C. For ATP assays, 50 μL of each thawed sample solution was added to 100 μL ice-cold Cell ATP-Releasing Reagent and incubated on ice for 5 min, followed by the addition of 100 μL ice-cold ATP Assay Mix (1:25 diluted in assay mix buffer). The reaction mixture was then incubated for 10 min in the dark at room temperature for the initial chemiluminescence flash period. The bioluminescence of each sample was measured with a high-sensitivity luminometer (Thermo Scientific).
4
Measuring ATP Levels in Oocytes
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The ATP concentration of oocytes was measured using a commercial assay kit (FL-ASC; Sigma). Briefly, oocytes were completely denuded of cumulus cells, and washed three times in PBS supplemented with 3 mg/ml polyvinylpyrrolidone.
Oocytes were transferred in groups of 10 to Eppendorf tubes containing 50 µl ice-cold PBS and stored at −80ºC until analysis. For analysis, samples were thawed on ice protected from light. Ice-cold somatic cell reagent (100 µl)
was added to each tube, briefly centrifuged, and incubated on ice for 5 min. Next, ice-cold assay mix solution (diluted 1:25 with ATP assay mix dilution buffer; 100 µl) was added, and each tube was briefly centrifuged and
incubated on ice for 5 min. Sample luminescence was measured in a flat bottom 96-well plate (Greiner Bio-One, Frickenhausen, Germany) using a microplate reader with a luminescence optical system (POLARstar Optima; BMG Labtech,
Ortenburg, Germany). A seven-point standard curve (0‒60 pM/tube) was included in each assay to enable the unknown ATP concentrations to be determined. The ATP concentration for each sample was then divided by the number of oocytes
in the tube to obtain the final value (expressed as pM ATP/oocyte).
Oocytes were transferred in groups of 10 to Eppendorf tubes containing 50 µl ice-cold PBS and stored at −80ºC until analysis. For analysis, samples were thawed on ice protected from light. Ice-cold somatic cell reagent (100 µl)
was added to each tube, briefly centrifuged, and incubated on ice for 5 min. Next, ice-cold assay mix solution (diluted 1:25 with ATP assay mix dilution buffer; 100 µl) was added, and each tube was briefly centrifuged and
incubated on ice for 5 min. Sample luminescence was measured in a flat bottom 96-well plate (Greiner Bio-One, Frickenhausen, Germany) using a microplate reader with a luminescence optical system (POLARstar Optima; BMG Labtech,
Ortenburg, Germany). A seven-point standard curve (0‒60 pM/tube) was included in each assay to enable the unknown ATP concentrations to be determined. The ATP concentration for each sample was then divided by the number of oocytes
in the tube to obtain the final value (expressed as pM ATP/oocyte).
5
Quantification of Protein and ATP
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