Spectramax m series multi mode microplate reader

To determine the IgM level of each patient, the complete dengue virus was tested by indirect enzyme-linked immunosorbent assay (ELISA). Briefly, different dengue recombinant envelope protein domain IIIs (rED III) antigens, derived from the four DENV serotypes, were coated on the bottom of 96-well polystyrene plate (NUNC, Pasadena, TX, USA) in 50 mM of bicarbonate buffer pH 9.6 at 4 °C overnight. The plate was washed with PBS-T and then blocked with 5% non-fat milk at 37 °C. After washing, human sera patient samples diluted (1:100) in blocking buffer were added to each well and incubated at 37 °C for 3 h. The samples were then washed and incubated with secondary antibody HRP-conjugated anti-human IgM, according to the manufacturer’s protocol. Stringent washing with PBS-T was performed five times to remove all nonspecific binding and 3,3′5,5′-tetra methyl benzidine (TMB) substrate solution (Invitrogen) was added to each well and the plates were kept in dark for 15 min. Finally, the reaction was stopped by 0.18 M sulfuric acid. Optical density (OD) was determined using the SpectraMax® M Series Multi-Mode Microplate Readers (Molecular Devices, San Jose, CA 95134, USA) at 450 nm.

Cells were lysed by RIPA buffer and sonication on ice. The deionized distilled water was added to make a final volume of 200 μl. The samples were centrifuged at 10000 rpm for 5 min, and then the supernatant was mixed with Bio-Rad protein assay dye reagent (Bio-Rad) for Bradford protein-binding assay. Absorption spectrum at 595 nm was detected for protein quantification (SpectraMax M series Multi-Mode Microplate Readers, Molecular Device).

Cell viability was measured using EZ-cytox water-soluble tetrazolium salt (Daeilbiotech, Suwon, Korea) according to the manufacturer’s manual. Cells (10⁴ cells per well of a 96-well plate) were seeded and incubated for 14 h. Cells were treated with various concentrations of tested chemicals for 18 h. A 10-μL aliquot of a detection reagent in the kit was added into each well (100 μL) and incubated for 1 h for 37 °C The absorbance at 450 nm was measured using a SpectraMax M Series Multi-Mode Microplate Readers (Molecular Devices, San Jose, CA, USA) at the Fluorescence Core Imaging Center.

A total of 5 × 10⁴ RAW264.7 cells were seeded into each well of XF-24 cell culture microplate (Seahorse Bioscience) and allowed to adhere. The cells were then supplemented with 50 μM PG(18:1)₂ or PG(18:2)₂ twice at 0 and 12 hour. At 24 hour, 100 ng/ml KLA was added and the cells were incubated for another 24 hours. The media was replaced with DMEM pH 7.4 for 1 hour at 37 °C. The cell oxygen consumption rate (OCR) was analyzed by Seahorse XFe Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA). The optimized concentrations of the mitochondrial inhibitors in the experiments were 10 μM oligomycin, 1 μM FCCP and 5 μM rotenone.
After seahorse mitochondrial assay, the protein concentration in each well of the XF24 cell culture microplate was measured by Bradford protein assay. The microplate was added 50 µl of 0.1% triton in PBS to each well and placed on ice for 5 min, and then 450 µl of DDW was added to each sample. The Bradford assay was started by mixing 10 µl of the prepared sample with 190 µl of 1X solution of Bradford protein-binding assay (Bio-Rad) in a 96-well plate. The absorbance at wavelength 595 nm of the plate was measured by SpectraMax M Series Multi-Mode Microplate Readers (Molecular Devices). The protein concentration was calculated based on the standard curve of BSA.