Synergy ht multi mode microplate reader

MCF-7 cells were seeded (2x10⁵ cell/mL) in 96-well plates for 24 h at 37°C in 5% CO₂. After 24 h, MCF-7 cells were treated with govaniadine (1 and 2 μM) in triplicate and incubated for 24 h. Control cells were treated with 0.1% DMSO. An assay for the intracellular conversion of nitro blue tetrazolium (NBT) to formazan by superoxide anion was used to measure the generation of reactive oxygen species in cells [18 (link)]. NTB was subsequently added at a final concentration of 1.2 mM to the wells and incubated in the dark for 1 h at 37°C. The formazan content of the cells was then solubilized with 100 mL of DMSO, and the absorbance was measured at OD 630 nm (Synergy™ HT Multi-Mode Microplate Reader).

Mouse serum samples were analyzed to quantify the amount of IL6 and GM-CSF (Mab Tag GmbH) using an immune-assay ELISA. The absorbance was measure at 540 nm using Synergy HT-Multi-Mode Microplate Reader.

ThT fluorescence assays were conducted to detect fibril formation in the fragmented peptides. A 1 mM protein solution was prepared using 100% HFIP for each species. In a non-treated 96-well microplate with a transparent bottom, 50 µL of the protein solution (final concentration 500 µM) and 50 µL of Tris-hydroxymethyl-aminomethane (Tris Buffer, pH 8) (final concentration at 25 mM) were dispensed. Each peptide was replicated into three repeated wells. ThT was added last at a concentration of 100 µM. To establish the background signal, a negative control was prepared and contained Tris buffer and ThT. The Synergy HT multi-mode microplate reader, set at an excitation wavelength of 440 nm and an emission wavelength of 485 nm, was used to measure the ThT fluorescence after the plate was sealed. Prior to each reading, the plate reader was slowly shaken for ten seconds. The fluorescence measurements were recorded every hour for duration of five days, and the procedure was repeated to ensure consistency. Statistical analysis of results involved calculating the average of the last five data points after subtracting the background signal.

Assays were carried out essentially as described⁴⁶ . Triplicate overnight cultures were diluted 1:100 into fresh, pre-warmed LB broth supplemented with appropriate antibiotics and grown to mid-exponential phase. 100 µl aliquots of each mid-log culture were mixed in with 900 µl Z-Buffer (0.85% w/v Na₂HP0₄, 0.55% w/v NaH₂PO₄, 0.07% w/v KCl, 0.025% w/v MgSO₄) to which 2.7 µl of 2-Mercaptoethanol (0.05 M) was added and the cell lysed by the addition of 40 µl choloroform, 20 µl 0.01% (w/v) SDS. After lysis 35 µl O-nitrophenyl-β-D-galactoside (ONPG: 4 mg/ml in Z-Buffer) was added to 176 µl of sample and the reaction proceeded in the dark at 28 °C until a yellow color developed when the reaction was terminated by the addition of 88 µl 1 M Na₂CO₃ to all wells. The plates were then read by measuring the OD_420nm in plate reader (Synergy™ HT Multi-Mode Microplate Reader) for each reaction and the β-galactosidase activity in Miller units determined using the equation: $$\mathrm{\beta }-\mathrm{galactosidase}\mathrm{activity}={\mathrm{OD}}_{420\mathrm{nm}(\mathrm{test})}-{\mathrm{OD}}_{420(\mathrm{blank})}/{\mathrm{OD}}_{600}\mathrm{\times }\mathrm{T}\mathrm{\times }\mathrm{V}$$ where T = time (min), V = volume (ml), and 1 Miller Unit is equivalent to the amount of enzyme which produced 1 µmol O-nitrophenol/min.

The green fluorescent protein (GFP) gene was amplified by PCR from pIJ-Potr using primer pair GFP-F and GFP-R (Wang et al., 2016 (link)). The PCR product was digested with SpeI and EcoRI, and then inserted into the corresponding sites of the integrative plasmid pSET152 to generate pSET152-GFP. The promoter region of glp operon (Pglp) was amplified with primer pair Pglp-F and Pglp-R using S. clavuligerus NRRL 3585 genomic DNA as the template. The PCR product was digested with BamHI and SpeI, and then inserted into pSET152-GFP. The resultant plasmid pSET152-Pglp-GFP was introduced into S. clavuligerus NRRL 3585 and ΔgylR mutant strains through conjugation to obtain WT-Pglp-GFP and ΔgylR-Pglp-GFP, respectively. As a control, the plasmid pSET152-GFP was transformed into S. clavuligerus NRRL 3585 to generate the recombinant strain WT-GFP. The above recombinant strains were cultivated in SA medium for 12 h. Then, 200 μl of cell culture was washed twice with pure water, and transferred into 96-well plates. The fluorescence intensities were recorded using a Synergy^™ HT Multi-Mode Microplate Reader with an excitation at 489 nm and an emission at 512 nm for analysis. Pure water was used as a blank control. The biomass of S. clavuligerus strains were detected by the simplified diphenylamine colorimetric method (Zhao et al., 2013 (link)).

ELISAs were performed to detect the presence of bovine IL-12 [25 (link)] and IFN-γ [26 (link)] as previously described. Absorbance was measured at 450 nm subtracted from 690 nm using the Synergy HT Multi-Mode Microplate Reader and Gen 5 software.