Synergy neo2 hts multi mode microplate reader

The plasma levels of semicarbazide-sensitive amine oxidase (SSAO) were determined using the Fluoro-SSAO assay Kit (Cell Technology, Inc. Hayward, CA USA, catalog # SSAO100-3). In this experiment, all plasma tested samples were diluted in ratio of 1:5 by reaction buffer. Since benzylamine served as a substrate for both SSAO and monoamine oxidase B, pargyline, a monoamine oxidase B inhibitor, was then added to a final concentration of 0.5 mM to each sample and incubated for 30 min at 37 °C. In a black 96-well plate, 100μL of standard or sample were added to each individual wells. Thereafter, 100μL of the reaction cocktail, a mixture of detection reagent, HRP and benzylamine were added to each well and incubated at 37 °C for 2 h. After 2 h, the plate was read with excitation at 530–570 nm and emission at 590 nm using Biotek Synergy Neo2 HTS Multi-Mode Microplate Reader.

Levels of human serum albumin-methylglyoxal adduct were measured in plasma samples using the competitive methylglyoxal (MG) ELISA kit (Hycult Biotech, Inc, Wayne, PA, USA, catalog # HIT503) based on the inhibition principle. In brief, plasma samples were thawed at room temperature and vortexed 30 s. Different standard concentrations and diluted plasma (1:5) were preincubated with labeled anti-MG trace antibody in U-shaped microtiter plate at room temperature. After 1 h, 100 μL of standard or samples mixed with tracer from the U-shaped plate were transferred in duplicate into appropriate wells in the coated microtiter plate coated with MG-adduct and incubated for 1 h at room temperature. After three washing, diluted streptavidin- Horseradish peroxidase (HRP) was added to each well and plate was incubated for 60 min at room temperature. Plate was washed three time and chromogenic substrate 3,3,5,5-tetramethylbenzidine (TMB) which catalyzes by HRP to generate a blue color was added. After 30 min, a 100 μL of acidic stop solution was added into each well and the absorbance at 450 nm was recorded using a using a Biotek Synergy Neo2 HTS Multi-Mode Microplate Reader (Männedorf, Switzerland). A standard curve was generated, and MG-adduct concentrations were calculated.

Fluid handling accuracy experiments were conducted by adding 1 µL water (at room temperature) and assessing level of replicate variance through mass measurement. The mass of an empty PCR tube was measured, 1 μL water was added with Opentrons, and the final mass was measured. The same operations were performed nine independent times using the same tip as well as with changing the tip at each step. To measure precision of automated CFE reaction preparation from same stock tubes of reagents, 1 μL of dye (food coloring) was added to 14 μL of water at room temperature. In total, 48 readings were taken using the same tip and changed tip at each step and they were done both manually and with the OT-2. The absorbance of each well was then measured using Synergy Neo2 HTS Multi-Mode Microplate Reader (BioTek) at 420 nm. Statistical assessment of variance was done with a two-sample F test. An F test can determine if the variance of one population is significantly different from variance of another and determine the associated P-value (35 ).

Analysis of ADCC was performed using an ADCC kit (Promega, cat. no. G7010) plus N1-transduced and nontransduced HEK 293 cells. For this purpose, cells were plated in triplicate (25 µl, 4 × 10⁵ cells/mL) in 96-well black walled plates (Corning) and then treated with serial 5-fold dilutions of zan-DNP in the presence or absence of 100-fold excess zanamivir. After incubating at rt for 30 min, 25 µl of 33.3 µg/mL human anti-DNP IgG1 (ACROBiosystems) were added to each well and the plates were incubated at rt for 30 min. Finally, ADCC effector cells were added at 75,000 cells/well and incubated for 6 h at 37 °C under 5% CO₂. The amount of firefly luciferase produced by ADCC effector cells was then quantified using Bio-GloTM Luciferase Assay Reagent (included in the kit). Luminescence was measure using Synergy Neo2 HTS Multi-Mode Microplate Reader (Biotek). % zan-DNP mediated ADCC was calculated as: luminescence _expt–luminescence _{no zan-DNP}.