ATP per optical density unit was quantified using Promega’s BacTiter-Glo kit coupled with a BioTek Synergy plate reader. BacTiter-Glo reagents and standards were prepared as described in the manual. Briefly, exponentially growing cultures of E. coli were treated according to the experimental parameters for the times indicated. When the time of treatment was reached 100 µL of culture, blank, or standard, was added to a black walled clear bottom 96-well plate. This was done in technical triplicate for each condition, blank, or standard, which had at least three biological replicates. Once the plate was prepared 100 µL of BacTiter-Glo Reagent was added to each well, and shaken in an orbital shaker for 1 min at room temperature, then left on the benchtop for 5 min. Luminescence was recorded using the BioTek Synergy plate reader, set to auto scaling and 1 s integration time. Simultaneously with BacTiter-Glo plate preparation, an optical density plate was created with the same cultures, and the absorbance of the culture was read on the same plate reader at 600 nm. ATP per OD unit was calculated by the average of the three biological replicates (which were averaged from the technical replicates), which were then divided by the obtained OD values.
Synergy plate reader
Manufactured by Agilent Technologies
Sourced in United States, United Kingdom, Switzerland, Germany
The Synergy plate reader is a multi-mode microplate reader designed for diverse applications in life science research. It is capable of performing absorbance, fluorescence, and luminescence measurements with high sensitivity and accuracy.
Automatically generated - may contain errors
Lab products found in correlation
Sytox green, by Thermo Fisher Scientific (3 mentions)
Synergy 2 plate reader, by Agilent Technologies (3 mentions)
Celltiter glo luminescent cell viability assay, by Promega (3 mentions)
Calcein am, by Thermo Fisher Scientific (3 mentions)
Pierce ecl, by Thermo Fisher Scientific (3 mentions)
Serum separation polymer, by BD (2 mentions)
Amylase detection reagent, by Horiba (2 mentions)
Microtainer vials, by BD (2 mentions)
Cytotox 96 non radioactive cytotoxicity assay kit, by Promega (2 mentions)
Matrigel, by BD (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
48 well plate, by Sarstedt (2 mentions)
Steady glo lysis luciferin reagent, by Promega (2 mentions)
Wizard genomic dna purification kit, by Promega (2 mentions)
Mouse insulin elisa kit, by ALPCO (2 mentions)
Kaleidagraph, by Synergy Software (2 mentions)
No taurocholate, by Merck Group (2 mentions)
Clear flat bottom 96 well plate, by Corning (2 mentions)
Sodium taurocholate, by Merck Group (2 mentions)
Epiquik m6a rna methylation quantitative colorimetric kit, by Epigentek (2 mentions)
177 protocols using synergy plate reader
1
Quantifying ATP Production in E. coli
2
Bile Tolerance of Lactobacillus Strains
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Overnight cultures were diluted 1:10 in MRS and loaded into a 96-well plate (100 µL per well). The CA/DCA bacteria were pre-exposed for 30 s to CA/DCA bile and washed 3 times with PBS prior to inoculation in MRS. For experiments using ox bile (Sigma-Aldrich, #70168, St. Louis, MO, USA), the bacterial cells were inoculated in pre-treated MRS at various concentrations. Then, 96-well plates were incubated for 24 h at 37 °C in a Biotek Synergy plate reader (Agilent, Santa Clara, CA, USA). During this incubation, optical density (600 nm) readings were taken every 5 min. Growth curve data were plotted and analyzed on GraphPad (Prism 8). These experiments were carried out in technical triplicates and repeated twice.
3
Biofilm Quantification with Safranin
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Overnight cultures were diluted 1:10 and incubated for 24 h in a 96-well plate (100 μL per well). Bacterial cells were inoculated in pre-treated MRS supplemented with either CA/DCA or ox bile. After 24 h of incubation, plates were washed with deionized water and air dried at 37 °C for 20 min. To stain the biofilm, safranin #65092B-95 purchased from Millipore Sigma (Darmstadt, Germany) was prepared (125 mL containing 6.04 g/L safranin, 19% ethanol, and 1% methanol, diluted in water) and added to the wells. After a 20 min incubation at room temperature, the safranin was discarded, and the wells were washed with deionized water and air dried at 37 °C. The stained biofilm was dissociated by resuspending in ethanol–acetone mix (80:20). The absorbance was read at OD490 nm in a Biotek Synergy plate reader (Agilent, Santa Clara, CA, USA). Results were normalized to blanks containing media only, graphed, and analyzed using GraphPad Prism (Prism 8). The experiments were performed as technical triplicates and in 3 biological replicates.
4
Protein Quantification of CeA Punches
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CeA punches were homogenized and assessed for protein concentration using a Pierce Bicinchoninic Acid (BCA) assay (Thermo Scientific, Rockford, IL) per manufacturer's protocol. Briefly, each sample was mixed with 100 μL of zirconium oxide beads (Next Advance, Inc., Troy, NY) and 200 μL of lab-made lysis buffer consisting of 137 mM NaCl, 20 mM Tris, 1% Igepal, 10% glycerol, and 1X Halt Protease Inhibitor Cocktail (Thermo Scientific, #87786). Samples then underwent mechanical disruption in a Bullet Blender (Next Advance Inc, Averill Park, NY) for 3 min on speed 8 at 4 °C followed by centrifugation at 1400 rcf for 15 min at 4 °C. The resulting homogenate for each sample was stored at −80° and a 12 μL aliquot was mixed in a 1:4 dilution with phosphate buffered sodium azide for BCA analysis. 25 μL of each sample or 25 μL of each albumin standard was pipetted in duplicate onto a 96-well plate. Working reagent was added at a volume of 200 μL to each well, and the plate was incubated at 37 °C for 30 min before reading at 562 nm on a Synergy plate reader (Agilent, Santa Clara, CA). Using the resulting protein concentration, homogenates containing 20 μg of protein were aliquoted and stored at −80 °C.
5
Cell Viability Assay Using MTT
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The cell metabolic activity was tested using the MTT assay. The cells were treated with HDACi concentrations ranging from 0.2 μm to 40 μm for 48 h in a 24 well plate. Afterward, cells were incubated with fresh medium, containing 0.5 mg/ml MTT (Sigma-Aldrich) for 1 h. The formazan crystals were dissolved by replacing the medium with DMSO/EtOH (1:2). The absorbance of the dissolved formazan crystals was measured at 570 and as a reference at 630 nm with a Biotek Synergy plate reader. R package “drc” was used to fit a dose-response curve and IC50 value was calculated based on the two-parameter log-logistic function (min: 0%, max: 100%) (21 ).
6
Proteasomal Chymotrypsin-like Activity Assay
7
Measuring Cell Viability with Inhibitor Treatments
8
Matador Assay for FfLuc Transfection
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Example 34
Development of Matador Assay Based on Transient Transfection of FfLuc in 293FT Cells
The mammalian expression vectors encoding Firefly luciferase cDNA (pLENTI-Blasticidin-FfLuc-A07; SEQ ID NO: 99) was transiently transfected into 293FT cells by calcium phosphate co-precipitation method. Approximately 18 hours post-transfection, one well for each transfection was treated for 90 min with Digitonin (final concentration 30 μg/ml) and the other well was left untreated. After the 90 minute incubation, 200 μl of supernatant was collected from each well in 1.5 ml tubes. The tubes were spun down at 1500 RPM for 5 minutes. Then, 25 μl of supernatant was collected and plated in a 384 well plate in triplicate (25 Luciferase activity was measured in well mode using BioTek synergy plate reader by addition to each well of 25 μl Luciferase assay buffer described in FfLuc Assay, which consisted per 1.0 ml of 885.5 μl assay buffer+1 μl DTT (1M stock)+100 μl of 10× Luciferin stock solution+13.5 μl ATP (100 mM stock). As shown in
9
Fluoride Sensitivity Assay in Yeast
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Confirmed plasmids were transformed into fex1Δfex2Δ yeast (strain SSY3 S1 File ) using the standard lithium acetate protocol. Transformed strains were selected on SD-ura plates at 30°C for 48 hr. For liquid growth assays, overnight cultures were diluted to a final OD600 of 0.1 in 24-well plate (Costar) in YPD with different amounts of NaF to a total volume of 1 mL per well. The assays were performed in a Biotek Synergy plate reader at 30°C with continuous agitation. Absorbance values were recorded every 3 min. Curves were plotted as absorbance change over time and analyzed using GraphPad Prism. To determine IC50 values, the area under the curve was calculated for each growth curve. These values were normalized relative to the growth curve without fluoride. The normalized values were plotted vs. the log of the fluoride concentration and fit to a standard dose–response curve with GraphPad Prism. The values are summarized in Tables 1 , 2 and 3 .
Yeast spot assay was performed from the overnight culture by adjusting OD600 = 1 and making a 10-fold dilution series in sterile water. Approximately, 5 μL of culture was plated on YPD or YPD + NaF agar plates and incubated for 48 h at 30°C before imaging.
Yeast spot assay was performed from the overnight culture by adjusting OD600 = 1 and making a 10-fold dilution series in sterile water. Approximately, 5 μL of culture was plated on YPD or YPD + NaF agar plates and incubated for 48 h at 30°C before imaging.
10
Mitochondrial Calcium Imaging in H4IIEC3 Cells
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The mitochondrial calcium indicator Rhod-2, AM (Invitrogen) was used to assess mitochondrial calcium in H4IIEC3 cells. Cells were pre-treated with indicated treatments and then loaded with 10 μM Rhod-2. Cells were loaded with the dye for 1 h, washed three times, and given fresh DMEM. Fluorescence was measured at ex/em of 552/581 using a Biotek Synergy plate reader.
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