Pr 4100 microplate reader

Cell viability was determined using CCK-8 assay. RAW264.7 mouse macrophage cells were inoculated in a 96-well plate culture for 2 h, then divided into different groups: (1) equal volume culture medium as the control group. (2) Equal volume LPS (1 μg/mL) treatment for 24 h as the model group. (3) Equal volume Gyp XVII and G-Rb1 with different concentrations (45, 90 and 180 µM) treatment for 24 h as the drug group. There were three replicates per group. After treatment, the cells were mixed with CCK-8 (10 μL) and incubated in the dark for another 4 h. The absorbance at 450 nm was recorded using a PR 4100 microplate reader (Bio-Rad Inc., Hercules, CA, USA).

Preliminary screening of the selected compounds was performed in 96-well plates using nitrocefin as a substrate. Final assay conditions include compounds (30 μM), NDM-1 (1 nM), HEPES (30 mM), ZnCl₂ (10 μM), NaCl (100 mM), BSA (20 μg/mL) at pH 6.8. EDTA (30 μM) was used as a positive control. After incubation at 30°C for 20 min, nitrocefin hydrolysis (100 μM) was monitored by following absorbance readings at 490 nm using a PR 4100 Microplate Reader (BIO-RAD; USA). The assay was performed in quadruplicate for all compounds and controls.