Spectramax id5 spectrophotometer

MC3T3-e1 cells were seeded in a 96-well plate at a density of 5000 cells/well and incubated for 24 h. The culture medium was then replaced with osteogenic induction medium with or without H₂O₂. The medium was changed every 2 days. After 7 days of osteogenic differentiation, ALP activity was measured using a TRAP and ALP assay kit (Takara, Shiga, Japan). The absorbance of each well was measured at 405 nm using a Spectramax iD5 spectrophotometer (Molecular Devices, Tokyo, Japan).

An indirect competitive ELISA was used to evaluate the ability of mAbs to disrupt peanut allergy serum IgE binding to Ara h 2. Microtiter plates were coated with 5 μg/mL natural Ara h 2 (Indoor Biotechnologies) overnight at 4°C and then blocked (PBST, consisting of 0.05% Tween-20 and 1% BSA) for 2 hours. Purified Ara h 2–specific mAbs (0.625–5 μg/mL) were added for 2 hours, followed by a 2-hour incubation with a 1:50 dilution of IgG-depleted peanut allergy pooled plasma from placebo-treated OIT participants (n = 6). For IgE detection, anti-IgE conjugated to HRP (1:10,000, Bethyl Laboratories) was added for 1 hour. Plates were washed with PBST between each step of the protocol, and all incubations were performed at room temperature, unless otherwise specified. For the colorimetric assay, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (BioLegend) was incubated for 45 minutes, and the reaction was stopped with 1 M phosphoric acid (Thermo Fisher Scientific). Absorbance was measured at 450 nm on a SpectraMax iD5 spectrophotometer (Molecular Devices) with SoftMax Pro 7.1 (Molecular Devices). Before analysis, the background was subtracted from OD values. The percentage of inhibition was calculated after normalization to the control (without a mAb).

P. aeruginosa ATCC 27853 was incubated with imipenem 1/4th MIC overnight to induce the expression of chromosomal β-lactamase in CAMH media. Cells were harvested, washed in 10 mM HEPES, 2.5 mM MgCl₂, pH = 7 buffer and resuspended in the same buffer at an OD₆₀₀ of approximately 0.5. Then, 100 μL of the cell suspension were mixed with 50 μL of either TXA01182 or polymyxin B to give a final concentration of 0 to 25 μg/mL, or 0 to 8 μg/mL, respectively. Next, 50 μL of nitrocefin was added to reach a final concentration of 32 μg/mL. Hydrolysis of nitrocefin was monitored spectrophotometrically by measurement of the increase in absorbance at 490 nm. Assays were performed in 96-well plates in a SpectraMax iD5 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).