The effects of human serum on the mitochondrial function of cultured cells was evaluated by measuring intracellular ATP content as described previously with minor modifications.5 (link) In short, we selected G418 resistant stable colonies from Hepa1c1c7 cells that had been transfected with pRL-TK using SuperFect (Qiagen, Germantown, MD, USA) for 3 weeks and stored. Cells showed stable Renilla luciferase activity. pRL-mTK-transfected mouse Hepa1c1c7 cells (5×104/well) in a 96-well plate were treated with 10 µL of heat-inactivated-serum samples for 48 h. The ATP content of the treated cells was determined using the luciferin-luciferase reaction with the CellTiter-Glo luciferase kit (Promega), with the output being normalized to Renilla luciferase activity. The intracellular ATP content of CSS-treated control cells was 65.1±2.7 nM. The ATP concentration of 10% sample serum-treated cells could be calculated from a standard curve of ATP concentrations (nM)=(% control+18.24)/1.817. ATP content was expressed as a percentage of CSS-treated control. The intra- and interassay coefficients of variation for these methods were <6.0%.
Celltiter glo luciferase kit
Manufactured by Promega
Sourced in United States
The CellTiter-Glo luciferase kit is a biochemical assay used to quantify the amount of ATP present in a cell sample. It provides a luminescent readout that is proportional to the amount of ATP, which is an indicator of metabolically active cells.
Automatically generated - may contain errors
4 protocols using celltiter glo luciferase kit
1
Measuring Mitochondrial Function in Cultured Cells
2
Measuring Mitochondrial Stress in Serum
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Levels of MIS in serum samples were evaluated by measuring intracellular ATP content (MIS-ATP) and ROS generation (MIS-ROS) as described12 (link),13 (link). pRL-mTK-transfected mouse Hepa1c1c7 cells (5 × 104/well) in a 96-well plate were treated with 10 μL heat-inactivated-serum samples for 48 h. The ATP content was determined using the CellTiter-Glo luciferase kit (Promega, Madison, WI, USA), with the output being normalized to Renilla luciferase activity. ROS level was determined using 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate and acetyl ester (CM-H2DCFDA; Molecular Probes, Eugene, OR, USA). Both MIS-ATP and MIS-ROS were expressed as % of charcoal stripped serum (CSS)-treated control. The intra- and inter-assay coefficients of variation for these methods were less than 6.0%.
3
Mitochondrial Inhibition by Serum Samples
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The mitochondrial inhibition induced by human serum samples was evaluated by determining intracellular ATP content [20 (link)]. In short, the Hepa1c1c7 cells (5×104/well) showing stable Renilla luciferase activity, were treated with 10% human serum samples or 10% CS-HS for 48 hours in a 96-well plate. The intracellular ATP content of the treated cells was determined using the luciferin-luciferase reaction and CellTiter-Glo luciferase kits (Promega) [29 (link)]. The measured ATP contents were normalized to Renilla luciferase activity, determined by adding an equal amount of Stop & Glo substrate solution of Dual-Glo Luciferase assay system (Promega). All data is presented as a percentage of control of ATP contents in 10% CS-HS-treated control cells. The intracellular ATP contents of the control cells were 65.1±2.7 nM. The ATP concentration of 10% sample serum-treated cells can be calculated from the standard curve of ATP concentration (nM)=(% Control+18.24)/1.817. The intra- and interassay coefficients of variation of these methods were less than 6.0%.
4
Evaluating Mitochondrial Inhibition by Serum
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The mitochondrial inhibition induced by serum samples was evaluated by measuring intracellular ATP contents9 (link),37 (link). Hepa1c1c7 cells were co-transfected with the pRL-mTK Renilla and pcDNA3.1 (neomycin +) plasmids using Superfect (Qiagen, Valencia, CA, USA). G418-resistant stable colonies were selected for 3 weeks, and then the clones showing stable Renilla luciferase activity were selected. Stable clones (5 × 104/well) were treated with serum samples of pregnant women (10 ul) or control serum in 96-well plates for 48 h. The intracellular ATP contents of the serum-treated cells were measured through the luciferin-luciferase reaction using CellTiter-Glo luciferase kits (Promega, Madison, WI, USA). Renilla luciferase activity was determined by adding an equal amount of Stop & Glo substrate solution from the Dual-Glo Luciferase assay kit (Promega, Madison, WI, USA). ATP concentrations were normalized to Renilla luciferase activity. The dose-dependent effects of four PCBs and TCDD on ATP concentration were also determined in the presence of control serum. The results were presented as % control of ATP content in control cells treated with control serum. Both intra- and inter-assay coefficients of variation were less than 6.0%.
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