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Synergy h1 hybrid plate reader

Manufactured by Agilent Technologies
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The Synergy H1 Hybrid plate reader is a versatile instrument used for multimode detection in life science research. It is capable of performing absorbance, fluorescence, and luminescence measurements on a variety of sample types in microplates.

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Lab products found in correlation

Fluo 8 calcium dye, by Abcam (3 mentions) Vybrant mtt cell viability assay, by Thermo Fisher Scientific (2 mentions) Celltiter glo luminescent cell viability assay, by Promega (2 mentions) Fluo 8 calcium dye, by Agilent Technologies (2 mentions) Lt1 transfection reagent, by Mirus Bio (2 mentions) Caspase glo 3 7 assay, by Promega (2 mentions) Tetracycline, by Thermo Fisher Scientific (2 mentions) Kanamycin, by IBI Scientific (2 mentions) Co no3 2 6h2o, by Merck Group (2 mentions) H3bo3, by Thermo Fisher Scientific (2 mentions) Cuso4 5h2o, by Merck Group (2 mentions) Opti mem, by Thermo Fisher Scientific (2 mentions) Polyethylenimine (pei), by Polysciences (2 mentions) Gene5 version 2, by Agilent Technologies (2 mentions) Passive lysis buffer (plb), by Promega (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Fs288, by R&D Systems (1 mentions) Fc actriib, by R&D Systems (1 mentions) Human il 6 elisa kit, by R&D Systems (1 mentions) Methyl thiazolyl tetrazolium (mtt), by Agilent Technologies (1 mentions)

61 protocols using synergy h1 hybrid plate reader

1

Inhibition of Cell Adhesion by SFTI-DBF Peptide

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Approximately 104 HFLS-RA cells per well were coated in 96 well plates, and cell adhesion assay was performed as described in our previous publication.14 (link) Different concentrations of SFTI-DBF were prepared in serum-free RPMI 1640 medium, and the cells were treated with 100 μl of different concentrations of peptides (0.00001–10 μM). Jurkat cells were fluorescently tagged by incubating it with [2′,7′-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxy-fluorescein, Acetoxymethyl Ester] (BCECF-AM) dye, then the fluorescently labelled Jurkat cells were incubated with peptide pretreated and non-treated HFLS-RA cells for 1 h. Then, the cells were washed, lysed, and the fluorescence was measured. The observed fluorescence was due to the adhesion of fluorescently labeled Jurkat cells to CD58 expressing OVCAR-3/HFLS-RA cells. The fluorescence reading was taken with a microplate fluorescence analyzer (Synergy H1 Hybrid plate reader Biotek, Winooski, VT, USA) with excitation wavelength at 485 nm and emission wavelength at 528 nm. A plot of percentage of inhibition of adhesion vs concetration was obtained to calculate the IC50 value.17 (link)
Dahal A., Parajuli P., Singh S.S., Shrestha L., Sonju J.J., Shrestha P., Chatzistamou I, & Jois S. (2022). Targeting protein–protein interaction for immunomodulation: A sunflower trypsin inhibitor analog peptidomimetic suppresses RA progression in CIA model. Journal of pharmacological sciences, 149(3), 124-138.
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Inhibition of Cell Adhesion by SFTI-DBF Peptide

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Approximately 104 HFLS-RA cells per well were coated in 96 well plates, and cell adhesion assay was performed as described in our previous publication.14 (link) Different concentrations of SFTI-DBF were prepared in serum-free RPMI 1640 medium, and the cells were treated with 100 μl of different concentrations of peptides (0.00001–10 μM). Jurkat cells were fluorescently tagged by incubating it with [2′,7′-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxy-fluorescein, Acetoxymethyl Ester] (BCECF-AM) dye, then the fluorescently labelled Jurkat cells were incubated with peptide pretreated and non-treated HFLS-RA cells for 1 h. Then, the cells were washed, lysed, and the fluorescence was measured. The observed fluorescence was due to the adhesion of fluorescently labeled Jurkat cells to CD58 expressing OVCAR-3/HFLS-RA cells. The fluorescence reading was taken with a microplate fluorescence analyzer (Synergy H1 Hybrid plate reader Biotek, Winooski, VT, USA) with excitation wavelength at 485 nm and emission wavelength at 528 nm. A plot of percentage of inhibition of adhesion vs concetration was obtained to calculate the IC50 value.17 (link)
Dahal A., Parajuli P., Singh S.S., Shrestha L., Sonju J.J., Shrestha P., Chatzistamou I, & Jois S. (2022). Targeting protein–protein interaction for immunomodulation: A sunflower trypsin inhibitor analog peptidomimetic suppresses RA progression in CIA model. Journal of pharmacological sciences, 149(3), 124-138.
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3

E. coli DNA Gyrase Supercoiling Assay

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DNA supercoiling assays were carried out in 30 μL of 1×gyrase buffer (35 mM Tris-HCl, 24 mM KCl, 4 mM MgCl2, 2 mM DTT, 0.1 mg/mL BSA, 6.5% glycerol, and 1.75 mM ATP, pH 7.5) containing 8.9 nM of E. coli DNA gyrase and 400 ng of relaxed plasmid pAB1 or pAB1_FL905. The reaction mixtures were incubated at 37°C for 30 minutes. For gel-based assays, the reactions were stopped by the addition of 1 μL of stop solution (2% SDS and 200 mM EDTA). DNA samples were analyzed by using 1% agarose gels in 1×TAE buffer followed by ethidium bromide staining and photographed under UV light. For SDFQ-based assays, DNA samples were transferred to a 384-well plate. Fluorescence measurements were performed in a Biotek Synergy H1 Hybrid Plate Reader using a wavelength of 494 nm and 521 nm for excitation and emission, respectively. For DNA gyrase inhibition assays, each compound was mixed with E. coli DNA gyrase and incubated on ice for 5 min. Then, relaxed pAB1 was added to initiate the supercoiling reaction.
Alfonso E.E., Troche R., Deng Z., Annamalai T., Chapagain P., Tse-Dinh Y.C, & Leng F. (2022). Potent inhibition of bacterial DNA gyrase by digallic acid and other gallate derivatives. ChemMedChem, 17(23), e202200301.
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4

Quantifying Cell Viability via MTT Assay

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A Vybrant MTT cell viability assay (Life Technologies) was used according to the manufacturer’s instructions. Briefly, 10 µl of 12 mM MTT (4,5-dimethylthiazol-2-yl-2-5-diphenyltetrazolium bromide) was added to 105 293T cells (different gene-edited lines, with or without WNV infection) in 100 µl of phenol-red free medium. Cells were incubated for 4 h at 37°C, at which time medium was removed and formazan crystals solubilized in 100 µl of DMSO were added for 10 min at 37°C. Liquid was analyzed for absorbance at 540 nm using a Synergy H1 Hybrid Plate Reader (Biotek).
Zhang R., Miner J.J., Gorman M.J., Rausch K., Ramage H., White J.P., Zuiani A., Zhang P., Fernandez E., Zhang Q., Dowd K.A., Pierson T.C., Cherry S, & Diamond M.S. (2016). A CRISPR screen defines a signal peptide processing pathway required by flaviviruses. Nature, 535(7610), 164-168.
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5

Ligand-Antagonist Assay for GDF8 and GDF11

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The assays using the HEK293-(CAGA)12 luciferase reporter cells (initially derived from RRID: CVCL_0045) were performed as previously described [47 (link)–50 (link)]. Cells were seeded in a 96-well plate and grown for 24 h. For the activity comparison assays, the growth medium was then removed and replaced with serum-free medium + 0.1% BSA containing a twofold serial dilution series of mature GDF8 or GDF11 for 18 h. Inhibition assays were performed in a similar fashion, except that the ligand was held at a final concentration of 0.62 nM and then mixed with twofold serial dilutions of antagonist (FS288, FSTL3, GASP1, GASP2, ActRIIB-ECD, Fc-ActRIIB; R&D Systems). The cells were lysed and luminescence was recorded immediately using a Synergy H1 Hybrid plate reader (BioTek). The activity data were imported into GraphPad Prism and fit using a non-linear regression with a variable slope to calculate the EC50 or IC50.
Walker R.G., Czepnik M., Goebel E.J., McCoy J.C., Vujic A., Cho M., Oh J., Aykul S., Walton K.L., Schang G., Bernard D.J., Hinck A.P., Harrison C.A., Martinez-Hackert E., Wagers A.J., Lee R.T, & Thompson T.B. (2017). Structural basis for potency differences between GDF8 and GDF11. BMC Biology, 15, 19.
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6

Broth Microdilution for Nisin MIC Determination

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The broth microdilution method described by Van Tassell et al., 2015 (link) was used to measure the minimal inhibitory concentration (MIC) of nisin for all strains of L. monocytogenes (wildtype, cheese outbreak isolates, and cell wall mutants), as well as the Pediococcus cerevisiae E66 (nisin sensitive strain) as a reference. Overnight cultures were inoculated at approximately 105 cfu/mL into 96-well microtiter plates containing serial 2-fold dilutions of nisin in BHI broth starting at 5 mg/mL. Plates were prepared in triplicate and OD600 measurements were made in a Synergy H1 Hybrid plate reader (BioTek, Winooski, VT, United States). The MIC was recorded as the lowest concentration of nisin that inhibited visible growth (Wiegand et al., 2008 (link)) after a 24 h incubation period at 37°C, and it is reported in Table 1.
Henderson L.O., Erazo Flores B.J., Skeens J., Kent D., Murphy S.I., Wiedmann M, & Guariglia-Oropeza V. (2020). Nevertheless, She Resisted – Role of the Environment on Listeria monocytogenes Sensitivity to Nisin Treatment in a Laboratory Cheese Model. Frontiers in Microbiology, 11, 635.
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7

Viral Envelope Extraction and Assay

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Isolated virus preparations were diluted to 1.0×108 PFU/ml (HSV-1) or 1.0×105 PFU/ml (PRV) in TNE in a final volume of 100 μl. Triton X-100 and NaCl were added to final concentrations of 2% and 100 mM, respectively, and incubated in the dark for 30 min (HSV-1) or 48 h (PRV) to extract viral envelopes. Samples were transferred to plates [CoStar, 3603] pre-mounted in a Synergy H1 hybrid plate reader [Biotek, Winooski, VT], and nitrocefin [Sigma Aldrich, 80017–706] was added at a final concentration of 0.1 mg/ml immediately before reading absorbance (HSV-1) or at the time of sample incubation (PRV). All experiments were replicated in triplicate.
Pegg C.E., Zaichick S.V., Bomba-Warczak E., Jovasevic V., Kim D., Kharkwal H., Wilson D.W., Walsh D., Sollars P.J., Pickard G.E., Savas J.N, & Smith G.A. (2021). Herpesviruses assimilate kinesin to produce motorized viral particles. Nature, 599(7886), 662-666.
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8

Quantifying Murine Plasma and Brain MMP-9

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We obtained murine MMP-9 detection kits from R&D System and analyzed levels of total MMP-9 in plasma and brains followed the manufacturer’s instructions. We recorded the results by a Biotek Synergy H1 Hybrid plate reader (wavelength = 450 nm).
Ren X., Hu H., Farooqi I, & Simpkins J.W. (2020). Blood substitution therapy rescues the brain of mice from ischemic damage. Nature Communications, 11, 4078.
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9

Quantifying Cell Viability via MTT Assay

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A Vybrant MTT cell viability assay (Life Technologies) was used according to the manufacturer’s instructions. Briefly, 10 µl of 12 mM MTT (4,5-dimethylthiazol-2-yl-2-5-diphenyltetrazolium bromide) was added to 105 293T cells (different gene-edited lines, with or without WNV infection) in 100 µl of phenol-red free medium. Cells were incubated for 4 h at 37°C, at which time medium was removed and formazan crystals solubilized in 100 µl of DMSO were added for 10 min at 37°C. Liquid was analyzed for absorbance at 540 nm using a Synergy H1 Hybrid Plate Reader (Biotek).
Zhang R., Miner J.J., Gorman M.J., Rausch K., Ramage H., White J.P., Zuiani A., Zhang P., Fernandez E., Zhang Q., Dowd K.A., Pierson T.C., Cherry S, & Diamond M.S. (2016). A CRISPR screen defines a signal peptide processing pathway required by flaviviruses. Nature, 535(7610), 164-168.
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10

Cell Viability Assay with CellTiter-Glo

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A CellTiter-Glo® Luminescent Cell Viability Assay (Promega) was performed according to the manufacturer’s instructions. Briefly, 2 x 104 of 3T3 or MEFs cells in 100 μl culture medium were seeded into opaque-walled 96-well plates. 24 h later, 100 μl of CellTiter-Glo® reagent was added to each well and allowed to shake for 2 min. After a 10 min incubation at room temperature for 10 min, luminescence was recorded by using a Synergy H1 Hybrid Plate Reader (Biotek) with an integration time of 0.5 second per well.
Zhang R., Kim A.S., Fox J.M., Nair S., Basore K., Klimstra W.B., Rimkunas R., Fong R.H., Lin H., Poddar S., Crowe JE J.r., Doranz B.J., Fremont D.H, & Diamond M.S. (2018). Mxra8 is a receptor for multiple arthritogenic alphaviruses. Nature, 557(7706), 570-574.
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