Dias microplate reader

The cytotoxicity of oxLDL was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. EPCs (2x10⁴ cells) were grown in 96-well plates and incubated with oxLDL at 0–100 μg/mL for 12 or 24 hours. Subsequently, MTT (0.5 μg/mL) was applied to cells for 4 h to allow the conversion of MTT into formazan crystals. After washing with PBS, the cells were lysed with dimethyl sulfoxide, and the absorbance was read at 530 nm with a DIAS Microplate Reader (Dynex Technologies, Chantilly, VA, USA).

The cytotoxicity of the recombinant GroEL protein was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Human PDL cells were grown in 96-well plates at a density of 1.6×10⁴ cells/well and incubated with serum-free medium (0 µg/mL; control), 25–100 µg/mL GroEL or 100 µg/mL GST for 24–72 h. Subsequently, 0.5 µg/ml MTT was added to each well, and incubation was continued at 37°C for an additional 4 h. Finally, the MTT solution was discarded, dimethyl sulfoxide (DMSO) was added to each well, and the absorbance was recorded at 530 nm using a DIAS Microplate Reader (Dynex Technologies Headquarters, Sullyfield Circle Chantilly, VA, USA). The effects of GroEL on cytokine production in PDL cells were measured by enzyme-linked immunosorbent assay (ELISA). Human PDL cells were seeded in 24-well plates at a density of 10⁶ cells/well and then treated with 0–50 µg/mL GroEL or 50 µg/mL GST in 500 µL serum-free medium for 24 h. The culture medium was collected for quantification of IL-6 and IL-8 levels using the DuoSet ELISA development kits (R&D Systems Inc., McKinley Place N.E., Minneapolis, USA.), and the absorbance was recorded using a TECAN Sunrise ELISA Reader (Tecan Group Ltd., Männedorf, Switzerland).