Varioskan flash spectral scanning multimode plate reader

Cellulase activity within the leachate was determined after opening the sample bottles. The mop materials were removed from the bottles, and the liquid was extracted from the top, middle, and bottom portions of each sample by inserting each portion into a sterile 30 mL syringe and squeezing liquid into sterile plastic tubes. The leachates were stored at -20°C until analyzed for cellulase activity. Cellulase activity was measured in the surrogate wastes using the fluorometric-based Cellulase Activity Assay Kit (Abcam, ab189817) that detects the release of a fluorescent compound, resorufin, from a cellulase substrate, resorufin-β-D-cellobioside (Coleman et al., 2007 (link)). Detection of resorufin was by measuring the emission at 595 nm after exciting the sample at 530 nm using a Varioskan Flash Spectral Scanning Multimode plate reader (Thermo Fisher). The detected cellulase activity was compared to the cellulase activity from T. reesei of 6.7 × 10^-4 μmole/mL/min (Coleman et al., 2007 (link)) as a reference. Samples having fluorescence values that exceeded the range of the standard curve were diluted and re-analyzed.

To detect Pol II phosphorylation under drug treatment, total proteins from GK cells were extracted with RIPA buffer (Millipore) and resolved on 6% SDS page. After transferring to PVDF membrane, Pol II was probed with antibody against total Pol II (clone N20, Santa Cruz, Dallas, TX), Pol II CTD P-ser2 phosphorylation clone H5 (abcam, Cambridge, UK), and Pol II CTD P-ser5 phosphorylation (abcam). For detecting IL-1B expression, 100 μl culture medium from each time points and treatments were collected and the antigen was detected with a rabbit anti-grouper IL-1β antibody prepared in-house [15 (link)]. The results of the ELISA assay were acquired using a Varioskan Flash spectral scanning multimode plate reader (ThermoScientific).

3T3-L1 preadipocytes were cultured in 96-well plates and induced to differentiate for 8 d. The cells were then starved overnight in DMEM with 0.1% BSA and further incubated in Krebs Ringer bicarbonate buffer (110 mM NaCl, 4.4 mM KCl, 1.45 mM KH₂PO₄, 1.2 mM MgCl₂, 2.3 mM CaCl₂, 4.8 mM NaHCO₃, 10 mM HEPES and 0.3% BSA) containing 100 µM NBD-glucose, 2.8 mM glucose and different concentrations of UA for 2 h at 37°C in the presence or absence of 1 µg/mL insulin. After 2 h incubation, the reaction was terminated by 20 µM of cytochalasin B. The cells were washed twice with cold Krebs Ringer bicarbonate buffer (4°C), and then 100 uL cold PBS (4°C) were added to each well. The fluorescence intensity was immediately measured at 466/550 nm on a Varioskan Flash spectral scanning multimode plate reader (Thermo Fisher Scientific, Waltham, MA). For the inhibition experiments, the cells were pretreated with PI3K inhibitor wortmannin (1 µM), MAPK inhibitor SB203580 (10 µM), and AMPK inhibitor compound C (2.5 µM), respectively, for 30 min.