Growth rates were estimated at different temperatures, pH, and NaCl concentrations. Only one parameter was changed each time and the two other parameter values were kept constant. Table 1 shows the different value combinations used. To prepare the preculture, approximately 20 mL of Thermus liquid medium were inoculated with strain OA30 and incubated overnight at 55 °C. The preculture was then transferred into a sterile 500 mL flask containing 100 mL of the same modified Thermus liquid medium to give an initial absorbance at 660 nm of at least 0.1. The culture was incubated in aerobic conditions using a Thermo Scientific MaxQ 4000 Benchtop Orbital Shaker (Thermo Scientific, Waltham, MA, USA) at 120 rpm for approximately 24 h. At different time intervals, the turbidity of the cultures was determined by measuring the increase in optical density at 660 nm with a Synergy H1 hybrid multi-mode microplate reader. At least 10 absorbance measurements were taken into account.
Synergy h1
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Synergy H1 is a multi-mode microplate reader designed for a wide range of applications. It features absorbance, fluorescence, and luminescence detection capabilities, allowing for flexible and versatile measurements across various experimental setups.
Automatically generated - may contain errors
Lab products found in correlation
Maxq 4000 benchtop orbital shaker, by Thermo Fisher Scientific (1 mentions)
Oil red o, by Thermo Fisher Scientific (1 mentions)
Oil red o, by Sinopharm (1 mentions)
Oil red o, by Merck Group (1 mentions)
Dcfh da assay kit, by Beyotime (1 mentions)
Sep pak c18 cartridge, by Waters Corporation (1 mentions)
Amicon ultra, by Merck Group (1 mentions)
Black polystyrene 96 well microplate, by Thermo Fisher Scientific (1 mentions)
6 protocols using synergy h1
1
Growth Kinetics of Thermus Strain OA30
2
Triglyceride Accumulation Visualization
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The accumulation of triglycerides on days 0, 3, 5, 7, and 10 in three groups was visualized by staining the cells with Oil Red O (Sigma). The cells were transplanted to a 24‐well plate, cultured in 37°C under 5% CO2 incubator for 2 hr, and then fixed with 10% formalin solution (Sinopharm Chemical Reagent Co.) for 20 min. The cells were stained with Oil Red O working solution for 10 min, rinsed with 60% isopropyl alcohol (Sinopharm Chemical Reagent Co.), and microphotographed. Oil Red O concentration was measured spectrophotometrically at 510 nm using a microplate reader (Synergy H1; BioTek, Thermo Fisher).
3
ROS Quantification in Kainic Acid Model
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A 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) assay kit (P0011, Beyotime Institute of Biotechnology, Shanghai, China) was used to detect the ROS level (Zhang et al., 2019 (link)) at 24 h and 3 days after KA administration. Animals were anesthetized using sodium pentobarbital (50 mg/kg, i.p.), and the cortex and hippocampus were separated quickly on ice. Then the samples (1 mg tissue added 10 μL 0.01 M PBS) were quickly cut with scissors on ice and filtered (200 mesh/inch stainless steel mesh) to prepare a single cell suspension. DCFH-DA was diluted with serum-free cell culture medium (1:1000; Beyotime, S0033, China) to a final concentration 10 μM/L. Then 500 μL DCFH-DA was added per 250 μL of single cell suspension and incubated for 40 min at 37°C in the dark. After repeated washing and centrifugation with 250 μL 0.01 M PBS, the fluorescence intensity of 200 μL samples were analyzed using a fluorescence microplate reader (Synergy H1; Thermo, United States; excitation and emission wavelength: 488 and 525 nm, respectively).
4
PEITC Entrapment Efficiency in Microparticles
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The PEITC entrapment efficiency (EE) was determined by ultra-filtration centrifugation [21 (link)]. 4 mL of MP suspension was placed in a centrifugal filter unit (MWCO 10,000, Amicon® Ultra, Millipore, MA, USA) and centrifuged at 8,000 rpm for 1 h to separate the un-entrapped PEITC. 3 mL of the filtrate containing the free PEITC was concentrated by solid-phase extraction (SPE), using C18 Sep-Pak® cartridges (Waters Corporation, Milford, MA USA) with elution in vacuo, using 500 μLpure methanol as elution solvent. Following, the absorbance of the eluates was measured at 245 nm. The same amount of the diluted MP suspension was dissolved in methanol at 60°C. The resultant sample was filtered through 0.45 μm-membranes and analysed for 245 nm absorbance. To determine the PEITC concentration, a calibration curve was constructed in methanol with PEITC concentrations of 0.2–6.6 mM. Measurements were made in 96-well Nunc UV transparent plate (Thermo Scientific, Waltham, MA, USA) with multidetection plate reader (Synergy H1, Vermont, USA) controlled by the Gen5 Biotek software version 3.04. The amount of PEITC entrapped in the MP was obtained by subtracting the free PEITC amount from the total PEITC amount in the suspension. The EE was calculated as follows:
5
Evaluating Gut Permeability in Rats
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To determine gut permeability, rats were starved overnight and then treated with BSA–FITC (INEP, Belgrade, Serbia) dissolved in water (oral administration, 100 mg/rat). The rats were left without access to water for 2 h, and then blood was collected. Serum was obtained by centrifugation at 2000 rpmi for 10 min in a MiniSpin centrifuge. Serum was pipetted in triplicate into 96 well optical bottom plates (Nunc), (50 μl/well) to measure fluorescence intensity (f.i.) using a Synergy H1 multi-plate reader (excitation at 485 nm/emission at 530 nm).
6
Oxygen Radical Absorbance Capacity Assay
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The oxygen radical absorbance capacity (ORAC) assay followed the protocol outlined by Coscueta et al. (2019) [52 (link)]. A black polystyrene 96-well microplate (Nunc, Roskilde, Denmark) was utilized, and measurements were taken using a Multidetection plate reader (Synergy H1, Vermont, USA) controlled by Gen5 Biotek software version 3.04. Fluorescence was monitored for 80 min at 1 min intervals. Each sample, standard, blank, or control analysis was duplicated. The final ORAC values were expressed as Trolox Equivalents (μmol TE).
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