Infinite 200 multimode microplate reader

The level of cellular ROS production was measured using the CellROX Green fluorescent sensor. Briefly, after seeding on 24-well plates at a density of 5 × 10⁴ cells/well, MOLM-14 and MV-4-11 cells were incubated in 10% FBS-supplemented RPMI media (1 mL) containing Gilt-MNC (800 nM) or free GLT at the same dose for 24 h. The CellROX Green reagent was then added to each well at a final concentration of 5 μM. After staining for 30 min at 37 °C, the cells were harvested in Eppendorf tubes and then centrifuged for 10 min at 8000 rpm at 4 °C. After discarding the supernatant, the cell pellet was rinsed with 1 mL of PBS (pH 7.4) twice to remove excess of the CellROX Green reagent. The resulting cells were dispersed in 100 µL of PBS (pH 7.4) and then transferred to each well of black-walled 96-well plates. The fluorescence intensity was recorded at an excitation wavelength of 485 nm and emission wavelength of 525 nm on a Tecan Infinite 200 multimode microplate reader. The cellular fluorescence images were obtained using an IX83 inverted microscope equipped with cellSens Dimension software (version 1.14, Olympus, Tokyo, Japan).

Cell viability was determined using the MTT assay. Cells were seeded on 96-well plates 24 h before treatment. The cells were then treated with various concentrations of DJC for 48 h, and then 20 μL MTT (5 mg/mL) was added to the culture medium in each well for 2–4 h until a purple precipitate was visible. Cells were then treated with 150 μL DMSO to dissolve the crystals and left at room temperature in the dark for 2 hours. The absorbance at 490 nm was recorded using a Tecan Infinite 200 Multimode microplate reader (Männedorf, Switzerland). Data are presented as a percentage of untreated control cultures.

NADPH oxidase activity was measured by lucigenin-enhanced chemiluminescence as previously described [20 (link)]. GMCs (5 × 10⁴ cells in 2 mL) were seeded on 12-well plates in quadruplicate and cultured with AGEs (250 μg/ml) for 48 h in the presence or absence of graded concentrations of DJC or AG490. After treatment, the cells were thoroughly washed by ice-cold PBS and resuspended in 500 μl of ice-cold hypotonic lysis buffer of pH 7.4 (20 mM Tris-HCl, 1 mM EDTA, and 1 mM EGTA and protease inhibitors). The cell suspensions were sonicated and centrifuged at 15,000 rpm for 10 min. The resulting pellets were resuspended and sonicated in lysis buffer. The supernatants were then centrifuged at 15,000 rpm for 60 min. The pellets containing whole cell membranes were resuspended in assay buffer (10 mM sodium phosphate, 2 mM potassium phosphate, 10 mM potassium chloride, 50 mM triethanolamine, 150 mM NaCl, 2 mM MgCl₂, 1 mM EDTA, and protease inhibitors) containing 10 μM lucigenin and 100 μM NADPH. The activity was measured using a Tecan Infinite 200 Multimode microplate reader (Männedorf, Switzerland). Values expressed as arbitrary light units (ALUs) per 100 μg protein were converted to the percent change from control. All of the protein concentrations were assayed using a BCA Protein Assay Kit.