In cell elisa kit

An equal number of L6 cells suspended in the growth medium was seeded on each well of a poly-L-lysine coated 96-well plate (Corning Inc. Corning, NY) and incubated overnight at 37°C in a CO₂ incubator. The cells were treated with different concentrations of LI80020F4 for 72 h, with a repeat treatment every 24 h in a fresh growth medium. The VC culture wells received 0.2% DMSO (v/v). Mitochondrial biogenesis was determined using an in-cell ELISA kit (Abcam, Cambridge, UK), following the assay protocol provided by the manufacturer. Briefly, the treated cells were fixed with 4% paraformaldehyde and probed with primary antibodies against COX-1 (cytochrome c oxidase subunit I) and SDHA. The PBS-washed cells were reacted with alkaline phosphatase (AP) and horseradish peroxidase (HRP)-labeled secondary antibodies (Jackson Immuno Research, West Grove, PA) for the detection of SDHA and COX-1 expressions at 405 and 600 nm, respectively, in a multi-mode plate reader (Spectramax M2e, Molecular Devices, Sunnyvale, CA). The ratio between COX-I and SDHA expressions was calculated, and the percent (%) increase of mitochondrial biogenesis was determined in the treated cells over the VC cells.

One hundred μl of the cell culture was transferred into a 96-well microplate provided by the In-Cell ELISA kit (Abcam, ab111541), and treated following the manufacturer’s recommendations. For detecting dsRNA, the mouse monoclonal antibody J2 (Scicons) diluted 1:400 was used as primary antibody. As secondary antibody, goat anti-mouse IgG (IRDye 800CW) diluted 1:1000 was used. The plate was stored at 4°C, and subsequently scanned on the Odyssey Infrared Imaging System using a fluorescent signal intensity, both 700 and 800 nm channels, and 4 mm focus. The fluorescence intensity of the image was analyzed on LI-COR Image Studio 4.0 Software for Odyssey. The microplate was normalized using the Janus green staining protocol as recommended by manufacturer instructions. Briefly, the microplate was emptied and 50 μl of 1 × Janus Green stain was added to every well for 5 min at room temperature, then the plate was washed 5 times with deionized water. Finally, 200 μl of 0.5 M hydrochloric acid was added to every well and incubated for 10 min. The microplate OD at 595 nm was measured in a microplate spectrophotometer SpectraMax iD3 (Molecular Devices).

An equal number of L6 cells suspended in the growth medium was seeded on each well of a poly-L-lysine coated 96-well plate (Corning Inc. Corning, NY) and incubated overnight at 37°C in a CO₂ incubator. The cells were treated with different concentrations of LI80020F4 for 72 h, with a repeat treatment every 24 h in a fresh growth medium. The VC culture wells received 0.2% DMSO (v/v). Mitochondrial biogenesis was determined using an in-cell ELISA kit (Abcam, Cambridge, UK), following the assay protocol provided by the manufacturer. Briefly, the treated cells were fixed with 4% paraformaldehyde and probed with primary antibodies against COX-1 (cytochrome c oxidase subunit I) and SDHA. The PBS-washed cells were reacted with alkaline phosphatase (AP) and horseradish peroxidase (HRP)-labeled secondary antibodies (Jackson Immuno Research, West Grove, PA) for the detection of SDHA and COX-1 expressions at 405 and 600 nm, respectively, in a multi-mode plate reader (Spectramax M2e, Molecular Devices, Sunnyvale, CA). The ratio between COX-I and SDHA expressions was calculated, and the percent (%) increase of mitochondrial biogenesis was determined in the treated cells over the VC cells.

An In-Cell ELISA Kit (MitoSciences Inc., Eugene, OR) was employed to evaluate the effect of simvastatin and control antibiotics (tetracycline and vancomycin) on mitochondrial protein synthesis and the experiment was conducted as described previously53 (link). The ratio between COX-I and SDH-A was calculated and the percent inhibition of mitochondrial protein synthesis was determined.