For in vivo deoxyviolacein quantification E. coli strain DH5 alpha pir were used. Plasmids were transformed and grown overnight as described for in vivo bulk fluorescence measurements. 150 μL of overnight culture was then removed and pelleted by centrifugation at 13,000 xg for 5 min. The pellet was resuspended in 150 μL of 100% ethanol (EtOH) and cells lysed by boiling for 5 min, followed by centrifugation at 13,000 xg for 5 min. One hundred μL of supernatant was then carefully removed, added to a costar 96-well plate and the deoxyviolacein quantified by measuring OD at 525 nm in a BioTek SynergyH1 microplate reader. OD 525 nm values of EtOH extracts from blank cells containing control plasmids were subtracted from values. Repeats and controls were as described for bulk fluorescence measurements.
Synergy h1 microplate
Manufactured by Agilent Technologies
Sourced in United States
The Synergy H1 microplate reader is a compact, multimode instrument designed for a variety of absorbance, fluorescence, and luminescence assays. It features a patented hybrid technology that combines monochromator and filter-based detection to provide flexibility and performance.
Automatically generated - may contain errors
Lab products found in correlation
Nuclear and cytoplasmic protein extraction kit, by Beyotime (1 mentions)
Hdac activity colorimetric assay kit, by Abcam (1 mentions)
96 well microtiter plates, by NEST Biotechnology (1 mentions)
Cell counting kit 8 (cck8), by Selleck Chemicals (1 mentions)
Thiazolyl blue mtt, by Merck Group (1 mentions)
Axioplan 2 light microscope, by Zeiss (1 mentions)
Adiponectin, by Abcam (1 mentions)
Tr22421, by Fujifilm (1 mentions)
Nefa hr, by Fujifilm (1 mentions)
Resazurin, by Merck Group (1 mentions)
Resazurin, by Agilent Technologies (1 mentions)
8 protocols using synergy h1 microplate
1
Quantification of Deoxyviolacein in E. coli
2
HDAC Activity Assay of ee-As4S4 in Kasumi-1 Cells
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The ee-As4S4 was prepared and characterized as described previously.18 (link) HDAC activity was detected according to the manufacturer’s protocol using a Colorimetric HDAC Activity Assay Kit (Bio Vision, San Francisco, CA, USA). Briefly, Kasumi-1 cells were collected and lysed with a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) after being treated with ee-As4S4. The nuclear protein of 85 μL (50 μg), assay buffer of 10 μL HDAC, and HDAC substrate of 5 μL were mixed and incubated at 37 °C for 1 h. Afterward, the reaction was stopped by adding 10 μL lysine developer and mixed well, and then incubated for 30 min at 37 °C. The absorbance was read at 405 nm with a Synergy H1 microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA).
3
Yeast Growth Kinetics on Sugars
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The growth profiles of Y. lipolytica strains growing on glucose, fructose and a mixture of both sugars were obtained using a Synergy H1 microplate reader (BioTek Instruments, Winooski, VT). Prior to the experiment, the cells were grown overnight in 5 mL YPD, washed thrice with sterile distilled water to remove any YPD residuals and standardized to OD600 = 10. The cultures were carried out in 96-well microtiter plates (NEST, Wuxi, China) with a working volume of 200 µL, 600 rpm linear shaking and initial OD600 of 0.5. The media consisted of 1.7 g/L YNB, 5 g/L NH4Cl, 50 mM phosphate buffer pH 6.8 and 10 g/L of glucose, or fructose. The temperature was maintained at 28 °C throughout the process. The growth was monitored by measuring optical density in 30 min intervals. Experiments were conducted in three biological replicates.
4
Evaluating AgNPs Toxicity with Antibiotics
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To evaluate the toxicity of AgNPs in combination with antibiotics, human fibroblasts (baby foreskin) were used. Cells were dispensed in 96-well microplates at a density of 5000 cells per well in DMEM supplemented with 1% fetal bovine serum (FBS) for 24 h at 37 °C. After 24 h the cells were incubated with the established concentrations of AgNPs and antibiotics for 24 h at 37 °C. Treatment was withdrawn and 100 μL MTT (3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (0.5 mg/mL) was added in DMEM without FBS for 4 h at 37 °C for the formation of formazan crystals. MTT was removed from the wells and 100 μL of DMSO was added to read absorbance in a Synergy H1 microplate reader with Gen 5 software (Biotek Instruments, Winooski, VT, USA) at a wavelength of 595 nm. As a positive control, cells were treated with hydrogen peroxide and as a negative control, they were only treated with medium. The assay was performed in triplicate.
5
Cell Viability Evaluation via CCK-8
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Cell viability assays were performed using a Cell Counting Kit-8 (CCK-8, Bimake, #B34302) according to the manufacturer’s instructions. The fluorescence intensity was measured in a Synergy H1 microplate reader (Biotek) at 450 nm. Wells containing only culturing medium served as controls.
6
MTT Assay for Cell Viability
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Thiazolyl blue (MTT, Sigma) reduction42 was used for the assessment of cell survival. Production of MTT formazan precipitates, dissolved in 100 µL DMSO per well, was measured as absorbance at 540 nm in a Synergy H1 microplate spectrofluorimeter (BioTek Instruments, Inc., Winooski, VT, USA). Cell survival was expressed as the percentage of absorption of treated cells in comparison with that of control cells (100% survival). The results presented were the mean value and SD from at least six independent experiments.
7
Comprehensive Metabolic Profiling in Liver Disease
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Fasting blood glucose was measured using a GLUCOCARD Vital glucometer (Arkay, MN USA). ELISAs measuring serum levels of Leptin (Crystal Chem 90,030), Adiponectin (Abcam ab108785), Insulin (ALPCO 80-INSMR-CH01), Transferrin (Abcam ab157724), Ferritin (Abcam, ab157713), Albumin (Abcam ab108791), and Hepcidin (Intrinsic LifeScience SKU# HMC-001) were quantified according to manufacturer’s protocol. Colorimetric assays measuring hepatic ALT activity (Sigma-Aldrich MAK052), serum triglycerides (ThermoFisher Scientific TR22421), and serum free fatty acids (Wako NEFA-HR (2)) were quantified according to manufacturer’s protocol. Hepatic iron content was quantified using an iron assay kit (Abcam ab83366), and hepatic triglycerides were quantified using the Non-Esterified Fatty Acid detection kit (Wako NEFA-HR (2)). All assays were performed in duplicate, and measured on a BioTek SYNERGY H1 microplate reader. A portion of liver was fixed in 10% formalin, embedded in paraffin, cut into 5 μm sections, and stained with Perls’ Prussian blue. Images were taken on a Zeiss Axioplan 2 light microscope.
8
Resazurin-based Cell Viability Assay
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20,000 CaSki cells per well were seeded in a 12-well plate. Drugs were added at 24 hours post doxycycline induction. At 72 hours after drug treatment, cells were incubated with fresh media containing 10 μg/ml resazurin (Sigma). resazurin conversion was measured with a Synergy H1 microplate reader (BioTek) with 560 nm excitation and 590 nm emission filters as previously described (80) .
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