96 well black bottom plate

Human ASCs were plated at 5 × 10³/cm² and differentiated in chemically defined medium for 28 days. Culture medium and lysed cell supernatant were individually collected on 14 and 28 days. Cells were lysed using 1% Triton-X solution (Sigma-Aldrich, St. Louis, MO). Cell culture lysate was collected by scrapping with a cell scraper (Fisher-Scientific) and briefly sonicated for 10 s using an ultrasonic water bath (Fisher-Scientific) on ice. Total secreted human adiponectin and leptin were measured using ELISA (R&D, Minneapolis, MN) per our prior methods2 (link)10 (link)11 (link)16 (link)17 (link). Absorbance was read using a microplate reader (Biorad, Hercules, CA). DNA content was measured using Quant-iT™ PicoGreen^® dsDNA Assay Kit (Life Technologies) per our prior methods. A high-range standard curve of 1 μg/mL was used for measuring the DNA concentration of the samples per our prior methods. The samples were prepared in a 96 well black bottom plate (Fisher-Scientific) with absorbance read using microplate reader and standard fluorescein wavelengths (excitation ~480 nm, emission ~520 nm; Molecular Devices, Sunnyvale, CA). Total triglycerides and glycerol contents were measured by processing the sample lysate with Free Glycerol Determination kit (Sigma-Aldrich) per our prior methods2 (link)10 (link)11 (link)16 (link)17 (link).

The levels of ROS were determined using a cellular assay probe 2,7-dichlorofluorescin diactate (DCFH-DA; Beyotime, Shanghai, China). Before ultrasound treatment, bacterial cells were incubated with DCFH-DA at a final concentration of 10 μM at 37°C for 20 min. Then, 200 μl of the sample was transferred into a 96-well black bottom plate (Fisher Scientific, Loughborough, Leicestershire, England) and measured using a fluorescent microplate reader at the excitation and emission wavelengths of 485 and 525 nm, respectively.

C. metallidurans CH34 was electroporated with the
plasmids for the riboswitch or constitutive promoter libraries. Cultures
were resuspended to OD₆₀₀ 0.05 in fresh LB media, and 200
μL was distributed in a black bottom 96-well plate (Thermo Scientific).
The plates were incubated in a Cytomat 2C incubator at 30 °C,
800 rpm for 48 h, and were fed into a Molecular Devices SpectraMax
i3 plate reader using a PreciseSCARA robotic arm OD₆₀₀.
mRFP1 was excited at 585 nm, emission was detected at 620 nm, and
OD measurement was done at 600 nm. mRFP1 levels (Arbitrary Units,
au) at the end of the experiment were plotted for each plasmid construct
for the constitutive promoter and riboswitch library. For the P_BAD_Riboswitch library, mRFP1 expression levels were studied
in the presence of 5 mM theophylline and 0.1% l-arabinose
(ON_ON) or in the presence of l-arabinose only (OFF_ON).
Activation ratios were calculated as follows A study of the relative contribution
of the
P_BAD promoter and RBI to the expression of mRFP1 was performed
without inducers (OFF_OFF), in the presence of only theophylline (Th_OFF),
only arabinose (OFF_Ar), and both inducers (Th_Ar)