Mithras lb 940 multimode microplate reader

Ten μL of the test sample was transferred into the wells of a 96-well plate, as well as the positive control (ciprofloxacin, stock 100 μg/mL) and blank (solvent) controls (DMSO and water). Each well of a microdilution plate was than inoculated with 190 μL of the diluted standardized inoculum (OD = 0.003 at 620 nm). Control wells were prepared with 190 μL MH broth and 10 μL extract to correct of any absorption due to extract components. The microdilution plates were placed in a shaker-incubator at 37°C for 18 h, and then read on a Mithras LB 940 Multimode Microplate Reader (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany) at 620 nm with a lamp energy of 13,000 using the MikroWin 2000 software package. The OD was measured at a wavelength of 620 nm and wells with a plant extract were corrected for the absorption contributed by the extract. Tests were typically carried out in duplicate. The relative inhibition (%) of the test sample was calculated by dividing the OD value of the test sample minus that of the non-inoculated extract control by the average OD of the solvent control, and multiplying by 100.

H. pylori gGT activity was measured using a colorimetric assay previously described^{27 (link)}. 2 × 10⁸ bacteria were resuspended in 1 ml of PBS and incubated at 37 °C (5% CO², 110 rpm) for 2 h. After centrifugation (13.000 rpm, 10 min, RT), 50 µl of supernatant per well were applied to a 96-well plate. Each well was filled up to a total volume of 200 µl per well with reaction buffer consisting of 5 mM L-gamma-glutamyl-p-nitroanilide (L-gGpNA) as substrate and 100 mM glycylglycine (Gly-Gly) as acceptor in 0.1 M Tris buffer, pH 8. Experiments were conducted in duplicates and performed three independent times. Absorption was monitored at 405 nm using a Mithras LB 940 Multimode Microplate Reader (Berthold Technologies) after 1 h of incubation at 37 °C.