Quantifying ATP Flux in C2C12 Cells

The ATP flux was examined by luminometry, as previously described^{5 (link),16 (link)}. C2C12 cells stably expressing pEF1, pEF1/Panx3, or Ser68Ala were seeded at 1.0 × 10⁴ cells/well in a 96-well plate, and cultured for 2 days in DMEM containing 10% FBS. The cells were then washed with PBS, followed by incubation in PBS for 2 min. The supernatant was collected and assayed with luciferase and luciferin (Promega). The luminescence was measured using a Mithras LB 940 multimode plate reader (Berthold).

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Ishikawa M., Williams G., Forcinito P., Ishikawa M., Petrie R.J., Saito K., Fukumoto S, & Yamada Y. (2019). Pannexin 3 ER Ca2+ channel gating is regulated by phosphorylation at the Serine 68 residue in osteoblast differentiation. Scientific Reports, 9, 18759.

Publication 2019

luciferase and luciferin Mithras LB 940 multimode plate reader

Cells Luciferase Luciferin Luminescence Promega

Corresponding Organization : Tohoku University

Other organizations : National Institutes of Health, National Institute of Dental and Craniofacial Research, Drexel University

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Variable analysis

independent variables

Expression of pEF1, pEF1/Panx3, or Ser68Ala in C2C12 cells

dependent variables

ATP flux measured by luminometry

control variables

C2C12 cell seeding density (1.0 × 10^4 cells/well)
Cell culture duration (2 days)
Cell culture medium (DMEM containing 10% FBS)
Incubation time in PBS (2 min)
Luminescence measurement using Mithras LB 940 multimode plate reader

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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