Synergy ht multi detection microplate reader

To test the dependence of G. murorum laccase activity on temperature, enzymatic assays were performed in a 96‐well flat‐wells plate (ThermoFisher Scientific) whose wells were filled with 398 μl of laccase reaction buffer (100 mM sodium acetate buffer, pH 4.5; 0.2 mM ABTS) used in triplicate for the test (enzymatic assays) and control (buffer along) samples. Once the plate was preheated in the plate‐reader (Synergy™ HT Multi‐Detection Microplate Reader; BioTek®; Agilent) to 20°C, 30°C, 40°C or 50°C, 2 μl of G. murorum protein sample (prepared as samples used for zymography) was added to the test samples and the absorbance of all samples was read for 20 min with 1 min intervals.

PDE activities were measured using a Total PDE Activity Assay Kit (Fluorometric) (BioVision, Cat No: K927-100). Briefly, differentiated adipocytes were extracted with PDE assay buffer on ice. The Coumarin Standard reagent was diluted serially from 5 to 40 pmol/reaction to establish standard curves. The Reaction Mix, which contains PDE substrates, was added to the samples, positive control and background control. The Sample Background Control Mix, which only contains sample extract and assay buffer, was added to the samples background control. The 96-well microplate was read at Ex/Em 370/450 nm on kinetic mode by using a SynergyTM HT Multi-Detection Microplate Reader (BioTek Instruments, Inc).

For each ATP and protein measurement a total of 3 co-cultured slices were placed in 400 µL Cell Lytic MT buffer (Sigma, Zwijndrecht, the Netherlands). These were homogenized twice (15 sec, 6500 g, 8°C) using a tissue homogenizer Precellys 24 Bertin Technologies (Labmakelaar Benelux B.V. Rotterdam, The Netherlands). To remove cellular debris, the homogenates were centrifuged for 5 min (14000 g, 8°C) and the remaining supernatant was divided into 2 portions of 200 µL. One portion was stored at −80°C for protein measurement and the second 200 µL portion was mixed with 100 µL of ATP lytic buffer from ATPlite kit (Perkin Elmer, Oosterhout, The Netherlands) for ATP measurement, which was carried out with a microplate reader Synergy TM HT Multi Detection Microplate Reader (Biotek Instruments Inc, Abcoude, the Netherlands) with settings for luminescence: 590/635 nm, top measurement, and sensitivity 230. ATP was determined in technical duplicates and luminescence values were recalculated as µM ATP in total liver slice extracts.
Protein concentration was determined by the Bradford method protein assay (BioRad, Veenendaal, The Netherlands). Protein samples of 2 µL were diluted 80 times in PBS and measured, with BSA used as a standard, each measurement being taken in duplicate. ATP concentration was normalized to mg of protein per slice.

The clonogenic assay for βPIX targeting shRNA transduced cells was quantified using crystal violet as previously described [25 (link)]. Briefly, following lentiviral transduction with pLKO.1 non-silencing and βPIX targeting shRNAs and puromycin selection, an equal number of cells were plated for each condition in a 24 well plate in triplicate. After 48 hours cells were washed twice with PBS and a mixture of 500 μL 6.0% glutaraldehyde and 0.5% crystal violet was added per well. Cells were incubated at RT for 30 min after which the solution was removed. The plates were rinsed with tap water and left to dry overnight. Visualization of the plates was obtained with an HP Scanjet 4850 scanner. Quantification was performed by dissolving the crystal violet stained cells in 300 μL PBS containing 1% sodium dodecyl sulfate (SDS) per well for 1 hr. at RT on a shaker. The absorbance was then read on a SynergyTM HT multi-detection microplate reader (BioTek Instruments) at 570 nm wavelength.

A modified version of the Bernfeld [22 (link)] protocol (dinitrosalicylic acid—DNS—method) was performed for the quantification of reduced sugars. In terms of reducing sugars, a calibration curve was determined using a D-Glucose solution with a concentration ranging between 0 and 2.5 g·L⁻¹ to estimate the amount of carbon present in the FW formulations. DNS reagent, containing 10 and 300 g·L⁻¹ of 3,5-dinitrosalicylic acid and potassium sodium tartrate, respectively, was applied, in equal volume, to the solubilized by-product samples. After addition, the mixtures containing the reagent were incubated at 100 °C for 5 min. Afterward, the mixtures were cooled by the addition of 5 mL of deionized water, and the absorbance (540 nm) was read in 96-well plates using a SynergyTM HT Multi-detection Microplate Reader (Bio-Tek Instruments, Inc., Winooski, VT, USA).

The specific lipid staining protocol was adopted to evaluate lipid accumulation in 3T3-L1 as previously described [60 (link)]. The 3T3-L1 cells were plated in 12-well plates, and adipocyte differentiation was induced with MDI solution for 8 days. During the adipocyte differentiation, the different concentrations of (−)-loliolide (0.062, 0.125, 0.25, 0.5, and 1 mM) were treated on days 0, 2, 4, and 6 except for the control group. On day 8, differentiated 3T3-L1 cells were fixed with 10% formalin for 1 h. Then, the fixed cells were washed twice with 60% 2-propanol and dried at 27 °C. The dried cell lipids were stained with 0.6% ORO solution for 2 h and again washed with distilled water (DW). After drying, ORO stain was eluted with 100% 2-propanol for 1 h in a shaking incubator. The relative ORO content was measured by Synergy^TM HT Multi-Detection Microplate Reader (Bio-Tek, Winooski, VT, USA). The images of the intracellular lipids from 3T3-L1 cells were captured using a microscope (Lionheart™ FX Automated Microscope, BioTek Instruments Inc., Winooski, VT, USA).