All LacZ assays were performed using strains containing a lacZ gene construct that was inserted into the endogenous lacZ gene in order to disrupt native lacZ expression. Bacterial samples were taken from back-diluted liquid cultures grown to late log phase (optical density at 600 nm [OD600], 1.0 to 1.5). Cell samples (200 μl) were loaded onto a clear 96-well plate, and OD600 measurements were taken using a Synergy 4 plate reader (BioTek). From these samples, 100 μl of cells was lysed for 25 to 35 min with a 10-μl solution containing PopCulture reagent (Novagen) and lysozyme (Thermo Fisher) in a 1,000:1 ratio. Samples (30 μl) of cell lysate were then incubated with 150 μl of o-nitrophenyl-β-d -galactopyranoside (ONPG) substrate solution (60 mM Na2HPO4, 40 mM NaH2PO4, 1 mg/ml ONPG, 2.7 μl/ml β-mercaptoethanol) in a 96-well plate at 28°C. The absorbance at 420 nm (OD420) was recorded every 30 s over 60 min by a Synergy 4 plate reader (BioTek). Final results were reported as the average slope (in mean OD420 per minute) of the 30-s intervals over the course of the 60-min incubation period, with the units reported as the LacZ activity (mean OD420 per minute per OD600). Statistical analysis was performed using GraphPad Prism (version 7) software.
Synergy 4 plate reader
Manufactured by Agilent Technologies
Sourced in United States
The Synergy 4 plate reader is a versatile instrument designed for a wide range of laboratory applications. It features optical modules that enable multiple detection modes, including absorbance, fluorescence, and luminescence. The Synergy 4 is capable of reading microplates with up to 384 wells, allowing for high-throughput sample analysis.
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Lab products found in correlation
Prism 8, by GraphPad (3 mentions)
Mtt assay, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Honeywell (3 mentions)
Wst 1 reagent, by Roche (3 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (3 mentions)
Amplex red reagent, by Thermo Fisher Scientific (2 mentions)
Superase in, by Thermo Fisher Scientific (2 mentions)
Gsh glo glutathione assay, by Promega (2 mentions)
Caspase glo 3 7 or 8 reagent, by Promega (2 mentions)
Half area high binding plates, by Corning (2 mentions)
Caspase glo 3 7 reagent, by Promega (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Alamarblue reagent, by Bio-Rad (1 mentions)
7 ethoxyresorufin, by AnaSpec (1 mentions)
Gas permeable microplate adhesive film, by Avantor (1 mentions)
Magmax rna isolation reagents, by Thermo Fisher Scientific (1 mentions)
Nano glo luciferase assay system, by Promega (1 mentions)
Luciferase assay system, by Promega (1 mentions)
Alamar blue dye, by Thermo Fisher Scientific (1 mentions)
Malachite green phosphate assay kit, by Cayman Chemical (1 mentions)
104 protocols using synergy 4 plate reader
1
LacZ Assay for Bacterial Cell Cultures
2
Caspase Activity Measurement in Cells
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From cell lysates: 25 μl of Caspase‐Glo®‐3/7 or ‐8 reagent (Promega, Southampton, UK) was added to 5μg (caspase‐3/7 activity) or 10μg (caspase‐8 activity) of cell lysate, made up to 25 μl with PBS in a white‐walled 96‐well plate. Each sample was plated in triplicate. The plate was incubated in the dark at room temperature for 45 min with gentle agitation. After incubation, the plate was read on a Synergy 4 plate reader (Biotek, USA) at a sensitivity of 200. Blank readings were taken in all cases to account for background luminescence. In live cells: Cells were seeded in triplicate at 5 × 103 per well in a white‐walled 96‐well plate. All drugging was performed in a total volume of 50 μl. After experimental procedures were completed, 50 μl Caspase‐Glo® reagent (Promega, Southampton, UK) was added to each well and the plate covered in foil to protect the reagent from ambient light. The plate was then incubated at room temperature for 45 min with gentle agitation. After incubation, the plate was read on a Synergy 4 plate reader (Biotek, USA) at a sensitivity of 200. Blank readings were taken in all cases to account for background luminescence.
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From cell lysates: 25 μl of Caspase‐Glo®‐3/7 or ‐8 reagent (Promega, Southampton, UK) was added to 5μg (caspase‐3/7 activity) or 10μg (caspase‐8 activity) of cell lysate, made up to 25 μl with PBS in a white‐walled 96‐well plate. Each sample was plated in triplicate. The plate was incubated in the dark at room temperature for 45 min with gentle agitation. After incubation, the plate was read on a Synergy 4 plate reader (Biotek, USA) at a sensitivity of 200. Blank readings were taken in all cases to account for background luminescence. In live cells: Cells were seeded in triplicate at 5 × 103 per well in a white‐walled 96‐well plate. All drugging was performed in a total volume of 50 μl. After experimental procedures were completed, 50 μl Caspase‐Glo® reagent (Promega, Southampton, UK) was added to each well and the plate covered in foil to protect the reagent from ambient light. The plate was then incubated at room temperature for 45 min with gentle agitation. After incubation, the plate was read on a Synergy 4 plate reader (Biotek, USA) at a sensitivity of 200. Blank readings were taken in all cases to account for background luminescence.
3
Cytotoxicity Assay for Claudin 18.2 Antibodies
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Example 71
5×104/well stably expressed Claudin 18.2 HEK293T cells and control HEK293T cells were seeded in tissue-culture flat-bottom microtiter plates. The next day growth medium was removed and the cells were incubated with 1:3 series dilution chimeric and humanized antibodies ch5C9 and h5C9o for 2 hours at 37° C., 5% CO2, 90% humidity. Human serum or plasma with complements was then added to a final concentration of 25% (v/v) and the cells were incubated at 37° C. for 3 hours. Cyto-Glo cytotoxicity assay reagent was added to each well, mixed and incubated at RT for 15 min. The luminescence signal was measured using a Bio-Tek plate reader synergy 4. EC50 was calculated through Prism Graphpad 8.0.1, nonlinear regression agonist vs response variable slope (four parameters). Results are shown in
4
Antibody-Mediated Cytotoxicity Assay Protocol
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Example 70
2.5×104/well 24 hour transiently expressed Claudin 18.2 HEK293T cells were seeded in tissue-culture flat-bottom microtiter plates. The next day growth medium was removed and the cells were incubated with 1:5 series dilution chimeric antibodies ch5C9, ch15G11 and ch9A1 for 2 hour at 37° C., 5% CO2, 90% humidity. Human serum or plasma with complements was then added to a final concentration of 25% (v/v) and the cells incubated at 37° C. for 3.5 hours. Cyto-Glo cytotoxicity assay reagent was added to each well. The plate was mixed and incubated at RT for 15 min. The luminescence signal was measured using a Bio-Tek plate reader synergy 4. EC50 was calculated through Prism Graphpad 8.0.1, nonlinear regression agonist vs response variable slope (four parameters). Results are shown in
5
Antibody-Mediated Cytotoxicity Assay
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Example 72
2×104/well stably expressed Claudin 18.2 HEK293T cells were seeded in tissue-culture flat-bottom microtiter plates. The next day growth medium was removed and the cells were incubated with 1:3 series dilution humanized antibodies h5C9o, h5C9ob, h5C9oap, h5C9oaq, h5C9oae, and h5C9oar for 2 hours at 37° C., 5% CO2, 90% humidity. Human serum or plasma with complements was then added to a final concentration of 25% (v/v) and the cells were incubated at 37° C. for 3 hours. Cyto-Glo cytotoxicity assay reagent was added to each well, mixed and incubated at RT for 15 min. The luminescence signal was measured using a Bio-Tek plate reader synergy 4. EC50 was calculated through Prism Graphpad 8.0.1, nonlinear regression agonist vs response variable slope (four parameters). Results are shown in
6
Serum Galectin-9 Quantification by ELISA
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The serum galectin-9 concentration was determined using a Quantikine ELISA Human Galectin-9 Kit (DGAL90 from R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Duplicates of serum samples were used at a dilution of 1:2. Plates were read in a Biotek Synergy-4 plate reader (Winooski, VT, USA) at 450 nm and 570 nm. To correct the optical imperfections of the plate, the values obtained from the readings at 450 nm were subtracted from the readings at 570 nm. An average of the duplicates of each sample was obtained, and the serum galectin-9 concentration determined by a standard curve was multiplied by the dilution factor.
7
Quantifying Cell Viability via AlamarBlue
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AlamarBlue® reagent (Bio-Rad, Hercules, CA, United States) was added to the cells at a volume equal to 1/10th of the culture medium volume. The cultures were returned to the incubator for a period of 1–2 h, depending upon cell type. A sample of the medium from each culture was transferred to a black-wall 96-well plate and fluorescence (545 nm excitation/590 nm emission) was read on a BioTek Synergy 4 plate reader (Winooski, VT, United States).
8
Cytochrome P450 Activity Assay
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CYP1A1 and CYP2C19 activities were measured fluorescently using ethoxyresorufin-O-deethylase (EROD) and 3-cyano-7-ethoxycoumarin (CEC) assays, respectively. For the EROD assay, cells were exposed to 7-ethoxyresorufin (10 μM, AnaSpec, Fremont, CA, United States) and salicylamide (inhibitor of phase II metabolism of resorufin, 1.5 mM, VWR) for 4 h. For the CEC assay, cells were exposed to CEC (25 μM) for 4 h. CYP1A2, CYP2B6, CYP2C9, and CYP3A4 activities were measured using CYP P450-GloTM assays (Promega) according to the manufacturer’s instructions. Omeprazole (20 μM) was used to induce CYP1A2. Phenobarbital (1 mM) was used to induce CYP2B6. Rifampin (10 μM) was used to induce all other CYP enzymes. Fluorescent and luminescent reads were performed on a BioTek Synergy 4 plate reader.
9
Bacterial Growth Kinetics Assay
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Growth studies of bacterial strains were conducted using microplate reader kinetic assays. Overnight cultures were inoculated into 10 ml of LB medium from single colonies and were grown at 30°C. These cultures were then washed twice with MOPS minimal media without any added carbon and diluted 1:100 into 500 μl of MOPS medium with a 10 mM concentration of a carbon source in 48-well plates (Falcon; catalog no. 353072). Plates were sealed with a gas-permeable microplate adhesive film (VWR, USA), and then optical density was monitored for 48 h using a BioTek Synergy 4 plate reader (BioTek, USA) at 30°C with fast continuous shaking. Optical density was measured at 600 nm, and all OD600 measurements are reported without path length corrections.
10
Bacterial Growth Kinetics Assay
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