Microplate reader

Cell proliferation was analyzed by a 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. Approximately 10⁴ cells per well were added into a 96-well cell culture plate. After the cells were cultured for 24, 48, 64, or 72 h, 50 μL of MTT (Sigma-Aldrich, USA) and 150 μL of the culture medium were added into each well of the cell culture plate. Then, the cell culture plates were incubated at 37 °C for 4 h. MTT was dissolved in 200 μL of DMSO in each well for 30 min. Finally, the absorbance of each well was measured at 570 nm on a microplate reader (BioAssay Systems, USA). All fold changes of cell proliferation were normalized according to the following formula: (Sample absorbance - Blank absorbance)/(Control absorbance - Blank absorbance). Each experiment was carried out in triplicate and repeated three times independently.

Plasma creatine kinase (CK) activity was measured as a marker of muscle fiber damage with a colorimetric method using a microplate reader (BioAssay Systems). Briefly, 10 μl plasma samples were added into separate wells in a 96-well plate, and 100 μl reconstituted reagents (10 μl substrate solution, 100 μl assay buffer and 1 μl enzyme mix) were added. Samples were incubated at 37 °C for 20 min and the plate was read at OD340nm at 20th min and 40th min.
Neutrophil Respiratory Burst (NRB) was measured with a neutrophil respiratory burst assay kit (Cayman) and data were collected by BD Accuri C6 flow cytometer. Briefly, 100 μl whole blood was added to a polypropylene tube containing 10 μl 10X dihydrorhodamine (DHR)-123. After incubation at 37 °C for 15 min, 25 μl 5X phorbol myristate acetate (PMA) was added and the assay mixture was incubated at 37 °C for 45 min. After addition of 2 ml red blood cell (RBC) lysis buffer, the tube was incubated at 37 °C for 10 min and centrifuged for 10 min at room temperature at 500×g. Supernatant was discarded and the cell pellet was resuspended in 0.5 ml assay buffer. DHR-123 was converted to a fluorescent compound rhodamine-123 by ROS, and the latter one emitted a green fluorescence (~ 530 nm) similar to fluorescein isothiocyanate (FITC) which was detectable in the FL-1 channel of the flow cytometer.