Synergy h4 hybrid plate reader

Manufactured by Agilent Technologies

Sourced in United States

The Synergy H4 Hybrid Plate Reader is a versatile instrument designed for a wide range of multi-mode detection applications in life science research and drug discovery. It features a combination of detection modes, including absorbance, fluorescence, luminescence, and time-resolved fluorescence. The Synergy H4 provides researchers with a flexible and reliable platform for various assays and experiments.

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Lab products found in correlation

Odyssey infrared laser scanner, by LI COR (2 mentions) 1 palmitoyl 2 oleoyl sn glycero 3 phosphocholine lipids, by Avanti Polar Lipids (2 mentions) 384 well flat clear bottom microplate, by Corning (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Exosome spin column, by Thermo Fisher Scientific (1 mentions) Igepal, by Merck Group (1 mentions) Anti nkp30 clone p30 15, by BioLegend (1 mentions) 5 5 dithiobis 2 nitrobenzoic acid dtmb, by Merck Group (1 mentions) Hepes, by Thermo Fisher Scientific (1 mentions) Thermomixer c, by Eppendorf (1 mentions) 2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Roche (1 mentions) E coli polar lipid extract, by Avanti Polar Lipids (1 mentions) Fluo 8 calcium flux assay, by Abcam (1 mentions) 384 black well polystyrene microplates, by Corning (1 mentions) Originpro 9, by OriginLab (1 mentions) Mtase glo reagent, by Promega (1 mentions) Mtase glo methyltransferase assay, by Promega (1 mentions) Opti minimal essential medium, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Synergy H4 Hybrid Reader Synergy H4 plate reader Synergy Hybrid plate reader Synergy H4 Hybrid Hybrid H4 Reader Synergy H4 reader H4 Synergy plate Synergy Hybrid Reader H4 plate reader Hybrid Plate Reader

21 protocols using synergy h4 hybrid plate reader

Saponin-Assisted Doxorubicin Encapsulation in IDEM and EXO

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Efficiently loading of doxorubicin within IDEM and EXO was achieved through saponin as an encapsulation enhancer. A 0.1% concentration of saponin was tested. Briefly, both nanoparticle solutions were mixed with 400 μg/mL of doxorubicin before adding saponin, and the mixture was incubated for 5 min at 37°C in agitation at 200 rpm. Consequently, unencapsulated doxorubicin was removed using an Exosome Spin Column (Invitrogen). After the loading, the encapsulation efficiency (EE%) was measured. To this, 0.1% of Triton-X-100 was added to the samples for 10 min at RT. The concentration of doxorubicin encapsulated in both IDEM (IDEM-DOXO) and EXO (EXO-DOXO) was determined by measuring its excitation and emission values (480 and 610 nm, respectively) against a set of known standards with a Synergy H4 Hybrid Plate Reader (Biotek).

Pisano S., Pierini I., Gu J., Gazze A., Francis L.W., Gonzalez D., Conlan R.S, & Corradetti B. (2020). Immune (Cell) Derived Exosome Mimetics (IDEM) as a Treatment for Ovarian Cancer. Frontiers in Cell and Developmental Biology, 8, 553576.

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NK cell cytotoxicity assay

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96-well flat-bottomed plates were pre-coated overnight at 4°C with 5 µg/ml anti-NKp30 (clone P30-15, Biolegend) and -CD18 (clone IB4) or with mouse IgG1aκ as an isotype control. Wells were blocked with phenol red-free RPMI complete medium then washed three times with PBS. 10⁵ WT or CD56-KO NK92 cells were added per well. Plates containing cells were briefly centrifuged then incubated for 90 min at 37°C 5% CO₂. Following incubation 1% v/v IGEPAL Sigma-Aldrich) was added to maximum release wells and plates were centrifuged at 1000 rpm. Supernatant was transferred and substrate solution containing PBS, HEPES (Gibco), N-α-Cbz-L-lysine thiobenzyl ester hydrochloride (BLT; Sigma-Aldrich) and 5,5’-Dithiobis(2-nitrobenzoic acid) (DTMB; Sigma-Aldrich) was added. The plate was incubated at 37°C 5% CO₂ for 30–60 min and luminescence was read at 415 nm using the BioTek Synergy H4 Hybrid plate reader. % maximum activity was calculated as: (sample absorbance – average background)/(average total release – average background) x 100%.

Gunesch J.T., Dixon A.L., Ebrahim T.A., Berrien-Elliott M.M., Tatineni S., Kumar T., Hegewisch-Solloa E., Fehniger T.A, & Mace E.M. (2020). CD56 regulates human NK cell cytotoxicity through Pyk2. eLife, 9, e57346.

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Quantifying Hemozoin Inhibition Activity

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An in vitro assay developed by Sandlin et al. was used to test for β-hematin, abiological hemozoin, inhibition activity (Sandlin et al., 2011 (link)). Test compounds were screened between 0.44 and 110 μM with a final DMSO concentration of 0.25% in a clear 384-well flat bottom microtiter plate (Corning). Positive controls of 80 μM amodiaquine and negative controls of 0.25% DMSO were used to evaluate the validity of the assay. Following the addition of each compound, 20 μL of water and Nonidet P-40 detergent (30.5 μM final concentration) were added to each well to mediate crystal formation. A 25 mM hemin chloride stock was prepared in DMSO and was passed through a 0.22 μm PVDF membrane filter. From this stock solution, a 228-μM hematin suspension was prepared in 2 M acetate buffer (pH 4.8) and added to the plate for a final hematin concentration of 100 μM. After a 6 h shaking incubation at 37 °C, pyridine was added to the plate (5% v/v final concentration) and was shaken an additional hour. The absorbance of the pyridine-ferrochrome complex was measured at 405 nm using a Synergy H4 Hybrid Plate Reader (BioTek). Dose response curves were generated from the maximum absorbance values using a nonlinear regression of sigmoidal dose response variable slope curve fit on GraphPad Prism v5.0.

Quiliano M., Mendoza A., Fong K.Y., Pabón A., Goldfarb N.E., Fabing I., Vettorazzi A., López de Cerain A., Dunn B.M., Garavito G., Wright D.W., Deharo E., Pérez-Silanes S., Aldana I, & Galiano S. (2016). Exploring the scope of new arylamino alcohol derivatives: Synthesis, antimalarial evaluation, toxicological studies, and target exploration. International Journal for Parasitology: Drugs and Drug Resistance, 6(3), 184-198.

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Spectrophotometric Analysis of μ-Oxo Heme Binding

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Spectrophotometric titrations were conducted to further understand the interaction of each compound with the μ-oxo heme dimer in vitro. First, each compound was dissolved in 40% aqueous DMSO and 0.02 M HEPES (pH 7.4) and serial diluted into a clear 96-well microtiter plate in triplicate. A hemin solution in the same solvent system was added to the plate to a final concentration of 5 μM. The plate was incubated for 1 h in the dark prior to measuring the absorbance at 400 nm using a Synergy H4 Hybrid Plate Reader (BioTek). Titrations were conducted without the heme addition to account for any absorbance from the compound itself at 400 nm and these values were subtracted from the final results. Absorbance values were plotted using a nonlinear least squares fit with CurveFit v1.00 to determine the equilibrium association constant.

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Fluorescence Polarization Assay for HalA2LP

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FP experiments were conducted as previously described^{58 (link)} with the following changes: HalM2 was serially diluted in GF buffer, then added to FP buffer (50 mM HEPES pH 7.5, 30 mM KCl, 5% glycerol, 1 mM MgCl₂, 1 mM TCEP, 0.25 mM AMP-PNP, 0.0025% IGEPAL-CA630, 10 nM Fluor-HalA2LP). The sample was allowed to incubate at RT for 15 min, then the parallel and perpendicular fluorescence intensities were measured. All experiments were conducted in triplicate in 386-well solid black polystyrene microplates (Corning) on a Synergy H4 Hybrid plate reader (BioTek). Data analysis was performed using Origin 9.6. Polarization was calculated using the following formula: P = (I_|| - I_⊥) / (I_|| + I_⊥). Competition FP assays were conducted using serially diluted HalA2LP analogs in GF buffer. Each dilution was mixed with FP Buffer + HalM2 (14 μM). Curves were fit to either non-linear dose-response or hyperbolic function. K_i was calculated using the following equation: K_i = IC₅₀ / (1 + [L]/K_d) where [L] = 10 nM Fluor-HalA2LP.

Rahman I.R., Acedo J.Z., Liu X.R., Zhu L., Arrington J., Gross M.L, & van der Donk W.A. (2020). Substrate Recognition by the Class II Lanthipeptide Synthetase HalM2. ACS chemical biology, 15(6), 1473-1486.

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Protein Concentration Measurement by BCA

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Protein concentration in the AH samples was measured while using bicinchoninic acid assay. Colorimetric reaction was monitored with Synergy H4 Hybrid plate reader (Biotek, Winooski, VT, USA). The calibration curve was constructed using 0.025–2 mg/mL bovine serum albumin solutions in PBS.

Chistyakov D.V., Baksheeva V.E., Tiulina V.V., Goriainov S.V., Azbukina N.V., Gancharova O.S., Arifulin E.A., Komarov S.V., Chistyakov V.V., Tikhomirova N.K., Zamyatnin AA J.r., Philippov P.P., Senin I.I., Sergeeva M.G, & Zernii E.Y. (2020). Mechanisms and Treatment of Light-Induced Retinal Degeneration-Associated Inflammation: Insights from Biochemical Profiling of the Aqueous Humor. International Journal of Molecular Sciences, 21(3), 704.

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Laccase Activity Assay with ABTS and Syringaldazine

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Laccase activity was assessed with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid; ABTS, Roche diagnostic, Mannheim, Germany) and syringaldazine (Thermo Scientific, Rockford, United States) as model substrates. As a reference, TvLac from Trametes versicolor (Sigma Aldrich, Saint Louis, MO, United States) was used. Enzymatic reactions were set up in 96-well plates with 200 μl total reaction volumes containing 0.1 mM substrate (ABTS or syringaldazine) in 50 mM Na-acetate buffer pH 5.0. The plate was incubated in an Eppendorf Thermomixer C (Eppendorf AG, Hamburg, Germany) at 37°C and 400 rpm and scanned with a Synergy H4 Hybrid plate reader (BioTek, Winooski, VT, United States) at 0.5, 5, 10, 20, and 30 min. Reactions were scanned at 350 and 740 nm for detecting ABTS and the ABTS radical, respectively, and at 530 nm for detecting the oxidized form of syringaldazine. In addition, the full spectrum of the final reactions, from 300 nm to 800 nm, was recorded after 31 min (see Supplementary Figure S1).

van Eerde A., Várnai A., Wang Y., Paruch L., Jameson J.K., Qiao F., Eiken H.G., Su H., Eijsink V.G, & Clarke J.L. (2022). Successful Production and Ligninolytic Activity of a Bacterial Laccase, Lac51, Made in Nicotiana benthamiana via Transient Expression. Frontiers in Plant Science, 13, 912293.

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AlphaScreen Assay for MUC1 Glycopeptide Binding

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Mixtures of His-tagged galectin-3
expressed in our laboratory^{24 (link)} (1.25 μL)
and biotin-ASF (1.25 μL) were added to the wells containing
varied concentrations (2.5 μL, final concentrations of 0–1
mM) of the MUC1 glycopeptide in assay buffer [25 mM Hepes, 100 mM
NaCl, and 0.05% Tween 20 (pH 7.4)]. The final concentration of His-tagged
galectin-3 was 200 nM and that of biotin-ASF 5 nM in the assay. The
reaction mixture was incubated for 1 h at room temperature; then 5
μL of nickel-chelate-Acceptor and 5 μL of streptavidin-conjugate
Donor beads were simultaneously added to a final concentration 25
μg/mL incubated for 1 h in dark at room temperature, and the
assay plate was subsequently read at 22 °C in the AlphaScreen
mode on the Synergy H4 Hybrid plate reader (BioTek). Data, AlphaScreen
signal counts (counts per second) versus log [inhibitor, M], were
expressed as means of five replicate measurements. The IC₅₀ values were obtained by nonlinear regression analysis using Graph
Pad Prism version 5.04.

Rodriguez M.C., Yegorova S., Pitteloud J.P., Chavaroche A.E., André S., Ardá A., Minond D., Jiménez-Barbero J., Gabius H.J, & Cudic M. (2015). Thermodynamic Switch in Binding of Adhesion/Growth Regulatory Human Galectin-3 to Tumor-Associated TF Antigen (CD176) and MUC1 Glycopeptides. Biochemistry, 54(29), 4462-4474.

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Real-Time Kinetic Assays for Rhomboid Proteases

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Real-time kinetic assays were performed as described (Dickey et al., 2013 (link)): 2-80 pmoles of rhomboid were mixed with 30 μg of liposomes formed from an E. coli polar lipid extract (Avanti Polar Lipids) and 25-1600 pmoles of FITC-TatA substrate in 50 mM Na-acetate, pH 4.0 and 150 mM NaCl. Proteoliposomes were formed by rapid dilution, collected by ultracentrifugation at 600,000g for 30 minutes, and proteolysis was initiated by neutralizing proteoliposomes with 50 mM Tris, pH 7.4, 150 mM NaCl (and simultaneously adding peptides without preincubation for inhibition experiments). Since membranes quench FITC-TatA, rhomboid-mediated proteolytic release was monitored at 37°C in real-time by quantifying FITC fluorescence in a Synergy H4 Hybrid plate reader (BioTek). For inhibition experiments, initial rates were extracted from progress curves monitored in real-time, reduction in rate was plotted as a function of inhibitor concentration, and modeled using the R environment with the equation:

\frac{v_{0, I}}{v_{0}} = \frac{K i}{K i + [I]}

in which v_0,I and v₀ are initial rates in the presence and absence of inhibitor, respectively.

Cho S., Dickey S.W, & Urban S. (2016). Crystal structures and inhibition kinetics reveal a two-stage catalytic mechanism with drug design implications for rhomboid proteolysis. Molecular cell, 61(3), 329-340.

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Reconstitution and Kinetics of Rhomboid Proteases

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Rhomboid proteases were co-reconstituted with substrates into liposomes using the inducible reconstitution system that we described recently^{16 (link)}. Bacterial rhomboid proteases were reconstituted in liposomes formed from an E. coli polar lipid extract, while DmRho4 was reconstituted in liposomes formed from a yeast polar lipid extract or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipids (Avanti Polar Lipids). All enzymes were assayed for 1 hour at 37°C, except DmRho4, which was assayed at 25°C for 2–4 hours. APP+Spi7-Flag reaction products were resolved by SDS-PAGE and quantified by anti-Flag western analysis using an Odyssey infrared laser scanner (LiCor Biosciences), while products for the real-time assay using FITC-TatA were quantified using a Synergy H4 Hybrid plate reader (Biotek) scanning once per minute. Proteolysis assays were supplemented with 0.5 mM calcium unless otherwise indicated. For calcium titration experiments, 10 pmol of DmRho4 and 200 pmol FITC-TatA were co-reconstituted into 30 µg yeast liposomes and total calcium was titrated from 0 to 1 mM. For kinetic analysis, 10 pmol of Rho4 was titrated against 15-600 pmol FITC-TatA substrate in the presence or absence of 0.5 mM calcium, initial reaction rates were extracted and fit to a Michaelis-Menten model using Prism software.

Baker R.P, & Urban S. (2015). Cytosolic extensions directly regulate a rhomboid protease by modulating substrate gating. Nature, 523(7558), 101-105.

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