Microplate reader

The supernatant of SV40-MES13 cells was collected at 48 h after treatment. The content of inflammatory cytokines in cell supernatant was detected using mouse tumor necrosis factor-alpha (TNF-α; cat. no. D721150), IL-1β (cat. no. D721017), IL-6 (cat. no. D721022), and IL-12 (cat. no. D721174) ELISA kits (Sangon Biotech Co., Ltd.), respectively. The experiment was performed in accordance with the manufacturer's protocol. The OD value at 450 nm was measured using a microplate reader (Tosoh Corporation).

Cell proliferation detection of SV40-MES13 cells was performed using Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Inc.). In brief, cells in the HG or LG group were plated on a 96-well plate (5x10³ cells/well) and transfected as indicated. At 0, 24, 48 and 72 h after transfection, 10 µl CCK-8 detection buffer was added and cells were incubated for 2 h at 37˚C. A microplate reader (Tosoh Corporation) was adopted to assess cell proliferation with an optical density (OD) value of 450 nm being measured.

The cell viability was evaluated using an alamarBlue assay (AbDSerotec, Oxon, UK) according to the manufacturer's instruction. The assay includes an indicator that fluoresces and undergoes colorimetric change when reduced by mitochondrial respiration, which is proportional to the number of living cells. The cells were cultured with DMEM containing 10% alamarBlue solution to evaluate the viability of the cells, and the cells were cultured for an additional 4.5 h. The absorbance in each well was measured using a micro plate reader (Tosoh Corp., Tokyo, Japan). The data are shown as values of Abs₅₇₀-Abs₆₀₀. Each experiment was repeated three times with six wells dedicated to each time point.