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Synergy 4 instrument

Manufactured by Agilent Technologies
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The Synergy 4 instrument is a multi-mode microplate reader designed for versatile and sensitive detection of various assays. It features robust optics and proprietary technologies to provide accurate and reliable data across a range of wavelengths and detection modes.

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Lab products found in correlation

Suc llvy amc, by Thermo Fisher Scientific (2 mentions) Z lle amc, by Thermo Fisher Scientific (2 mentions) Boc lstr amc, by Merck Group (2 mentions) Mg132, by Merck Group (2 mentions) Creatine kinase activity assay kit, by Merck Group (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Bay k8644, by Merck Group (1 mentions) Fluo 4 direct calcium assay, by Thermo Fisher Scientific (1 mentions) Fluo 4 direct calcium reagent, by Thermo Fisher Scientific (1 mentions) Prism 6, by GraphPad (1 mentions) Halotag nanobret 618 ligand, by Promega (1 mentions) Pfn21a halotag cmv flexi vector, by Promega (1 mentions) Probenicid, by Merck Group (1 mentions) Fluo 4 am, by Thermo Fisher Scientific (1 mentions) Halotag pfn21a, by Promega (1 mentions) Nanobret 618 ligand, by Promega (1 mentions) Dual glo luciferase assay, by Promega (1 mentions)

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Synergy 4 (661 citations)

10 protocols using synergy 4 instrument

1

Fluo-4 Direct Calcium Assay in HL-1 Cells

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The Fluo-4 Direct Calcium Assay was performed as described by the manufacturer (Thermo Fisher Scientific). Briefly, HL-1 cells, pretreated as described, were stimulated with Fluo-4 Direct calcium reagent (Thermo Fisher Scientific) and signals were detected one hour post treatment. 10 mM Bay K8644 (Sigma) was added to cells and signals were immediately detected using a Synergy 4 instrument (BioTek). Results were analyzed using Prism 6.0 software (GraphPad Software, CA).
Romanelli A., Affinito A., Avitabile C., Catuogno S., Ceriotti P., Iaboni M., Modica J., Condorelli G, & Catalucci D. (2018). An anti-PDGFRβ aptamer for selective delivery of small therapeutic peptide to cardiac cells. PLoS ONE, 13(3), e0193392.
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2

PALLD and ANKRD1/FHOD1 Interaction Study

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HEK293 cells cotransfected with pFN21A HaloTag CMV Flexi vector (Promega) expressing PALLD and pNLF1-N [CMV Hygro] or pNLF1-C [CMV Hygro] vector (Promaga) expressing ANKRD1 or FHOD1 were treated with 100 nM HaloTag NanoBRET 618 Ligand (Promega), whereafter signals were detected 6 hr later using a Synergy 4 instrument (BioTek). Results were analyzed using GraphPad Prism 9.0 software.
Mastrototaro G., Carullo P., Zhang J., Scellini B., Piroddi N., Nemska S., Filomena M.C., Serio S., Otey C.A., Tesi C., Emrich F., Linke W.A., Poggesi C., Boncompagni S, & Bang M.L. (2023). Ablation of palladin in adult heart causes dilated cardiomyopathy associated with intercalated disc abnormalities. eLife, 12, e78629.
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3

Calcium Imaging of GHRHR Cells

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GC-GHRHR cells, transiently transfected with wt-hGH and treated for 24 h with 5 mM butyrate, were plated in half-area clear bottom 96-well plates (Corning, CA, USA) at 753 cells/well. 24 h later, following two washings with OPTIMEM medium +2.5 mM probenicid (Sigma-Aldrich, Buchs, Switzerland), cells were incubated with 2 µg/mL Fluo-4 AM (Invitrogen, LifeTechnologies, Basel, Switzerland) and Pluronic F-127 0.025% (w/v) (Invitrogen, LifeTechnologies, Basel, Switzerland) for 30 min in dark at 37°C. After washing twice with OPTIMEM medium, cell measurement was performed on a Synergy-4 instrument (BioTek, Highland Park, VT, USA) with an excitation band of 485/20 nm and fluorescence was measured at 528/20 nm. Baseline signal (F0) was recorded during 5 min before the addition of each stimulus. Subsequently, continuous fluorescence measurements were performed for 20 to 30 min. Ionomycin (Calbiochem, San Diego, CA, USA) stimulation was used as a positive control. Results are shown as F/F0 ratios after background subtraction, where F was the fluorescence signal intensity and F0 was the baseline as calculated by averaging the ten frames before stimulus application.
For [Ca2+]i measurements in siRNA-treated cells, cells were further transfected with wt-hGH 48 h post-siRNA transfection and treated with 5 mM butyrate. After 24 h cells were seeded and treated as mentioned above.
Miletta M.C., Petkovic V., Eblé A., Ammann R.A., Flück C.E, & Mullis P.E. (2014). Butyrate Increases Intracellular Calcium Levels and Enhances Growth Hormone Release from Rat Anterior Pituitary Cells via the G-Protein-Coupled Receptors GPR41 and 43. PLoS ONE, 9(10), e107388.
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4

Proteasome Activity Assay in Liver

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For measuring proteasome activity in vitro from liver extracts we utilized fluorogenic model substrates as described (Cui et al., 2014 (link)). Briefly, liver was Dounce homogenized in proteasome reaction buffer (50 mM Tris, 1mM EDTA, 150 mM NaCl, 5 mM MgCl2, 0.5 mM DTT, pH 7.5) and clarified by centrifugation. 20 μg of liver protein was then incubated with 100 μM of Suc-LLVY-AMC (Fisher Scientific), Z-LLE-AMC (Fisher Scientific), or Boc-LSTR-AMC (Sigma) to measure chymotrypsin, caspase, and trypsin-like proteasomal activities, respectively, plus freshly added 0.1 mM ATP and 0.5 mM DTT. As a negative control duplicate samples were incubated with mg132 (Sigma) as described (Cui et al., 2014 (link)). The fluorescence accumulation was monitored for 2 hours at 37°C and an excitation/emission of 380/460 using a BioTek Synergy 4 instrument. Fluorescence was converted to nmol of AMC cleaved per minute per mL and normalized by dividing individual activities by the average assay activity.
Ryzhikov M., Ehlers A., Steinberg D., Xie W., Oberlander E., Brown S., Gilmore P.E., Townsend R.R., Lane W.S., Dolinay T., Nakahira K., Choi A.M, & Haspel J.A. (2019). Diurnal Rhythms Spatially and Temporally Organize Autophagy. Cell reports, 26(7), 1880-1892.e6.
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5

BRET Assays in HL-1 Cardiac Cells

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BRET assays were performed in HL‐1 cardiac cells as previously described (Rusconi et al, 2016). Briefly, Cavα1.2 and ACTN2 (WT and mutant) cDNAs were cloned into the pNLF1‐N and the HaloTag‐pFN21A vector (Promega), respectively, and then transfected in HL‐1 cells in a Cavα1.2‐NanoLuc:ACTN2‐Halo (WT or mutant) 1:100 ratio. Cavβ2 was added as indicated. Forty‐eight hours after transfections, cells were treated with 100 nmol/l NanoBRET 618 ligand, and signals were detected 5 h after treatment, with a Synergy 4 instrument (BioTek).
Prondzynski M., Lemoine M.D., Zech A.T., Horváth A., Di Mauro V., Koivumäki J.T., Kresin N., Busch J., Krause T., Krämer E., Schlossarek S., Spohn M., Friedrich F.W., Münch J., Laufer S.D., Redwood C., Volk A.E., Hansen A., Mearini G., Catalucci D., Meyer C., Christ T., Patten M., Eschenhagen T, & Carrier L. (2019). Disease modeling of a mutation in α‐actinin 2 guides clinical therapy in hypertrophic cardiomyopathy. EMBO Molecular Medicine, 11(12), e11115.
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6

Proteasome Activity Assay in Liver

Cited 1 time
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For measuring proteasome activity in vitro from liver extracts we utilized fluorogenic model substrates as described (Cui et al., 2014 (link)). Briefly, liver was Dounce homogenized in proteasome reaction buffer (50 mM Tris, 1mM EDTA, 150 mM NaCl, 5 mM MgCl2, 0.5 mM DTT, pH 7.5) and clarified by centrifugation. 20 μg of liver protein was then incubated with 100 μM of Suc-LLVY-AMC (Fisher Scientific), Z-LLE-AMC (Fisher Scientific), or Boc-LSTR-AMC (Sigma) to measure chymotrypsin, caspase, and trypsin-like proteasomal activities, respectively, plus freshly added 0.1 mM ATP and 0.5 mM DTT. As a negative control duplicate samples were incubated with mg132 (Sigma) as described (Cui et al., 2014 (link)). The fluorescence accumulation was monitored for 2 hours at 37°C and an excitation/emission of 380/460 using a BioTek Synergy 4 instrument. Fluorescence was converted to nmol of AMC cleaved per minute per mL and normalized by dividing individual activities by the average assay activity.
Ryzhikov M., Ehlers A., Steinberg D., Xie W., Oberlander E., Brown S., Gilmore P.E., Townsend R.R., Lane W.S., Dolinay T., Nakahira K., Choi A.M, & Haspel J.A. (2019). Diurnal Rhythms Spatially and Temporally Organize Autophagy. Cell reports, 26(7), 1880-1892.e6.
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7

Creatine Kinase Activity Assay

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Blood was collected from jugular veins before euthanasia. Blood was left for 15 min at room temperature and centrifuged (3500 g for 10 min at 4 °C) to promote clot formation and serum collection. CK activity was determined using the Creatine Kinase Activity Assay Kit (Sigma-Aldrich) and the Synergy 4 instrument (BioTek Instruments, USA).
Starikova A.V., Skopenkova V.V., Polikarpova A.V., Reshetov D.A., Vassilieva S.G., Velyaev O.A., Shmidt A.A., Savchenko I.M., Soldatov V.O., Egorova T.V, & Bardina M.V. (2022). Therapeutic potential of highly functional codon-optimized microutrophin for muscle-specific expression. Scientific Reports, 12, 848.
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8

NanoBRET Assay for Protein Interactions

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The NanoBRET Assay was performed as previously described (Rusconi et al., 2016 (link)). Briefly, transfected HEK293 cells were treated with 100 nM NanoBRET 618 Ligand (Promega), and signals were detected 6 h after treatment. Signals were detected using a Synergy 4 instrument (BioTek).
Di Mauro V., Ceriotti P., Lodola F., Salvarani N., Modica J., Bang M.L., Mazzanti A., Napolitano C., Priori S.G, & Catalucci D. (2021). Peptide-Based Targeting of the L-Type Calcium Channel Corrects the Loss-of-Function Phenotype of Two Novel Mutations of the CACNA1 Gene Associated With Brugada Syndrome. Frontiers in Physiology, 11, 616819.
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9

MCL1 3'UTR Luciferase Assay

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The pmirGLO plasmids containing wild-type (GLO-MCL1-UTR) or mutated (GLO-MCL1-muUTR) MCL1 3′UTRs, and vector control and miR-181c expressing plasmids were cotransfected using Lipofectamine 3000 (Thermo Fisher, Carlsbad, CA, USA) into HEK293 cells. A luciferase reporter assay was performed with the Dual-Glo Luciferase Assay (Promega). Cells were rinsed with PBS and lysed with the passive lysis buffer 48 h after transfection. A fraction of lysate was used for RNA isolation and the expression of miR-181c and 18S internal control were quantitated by real-time RT-PCR with assays from Thermo Fisher as described earlier. The remaining lysate was used for luciferase quantitation using the protocol as recommended by Promega. Firefly and renilla luciferases were quantitated using Synergy 4 instrument with Gen5 software (BioTek). Relative luciferase level was calculated as firefly luciferase level normalized to renilla luciferase control level.
Wang W., Chen R., Droll S., Barber E., Saleh L., Corrigan-Cummins M., Trick M., Anastas V., Hawk N.V., Zhao Z., Vinh D.C., Hsu A., Hickstein D.D., Holland S.M, & Calvo K.R. (2021). miR-181c regulates MCL1 and cell survival in GATA2 deficient cells. Journal of leukocyte biology, 111(4), 805-816.
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10

Blood Collection and CK Analysis

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Cardiac puncture under avertin anaesthesia was used to collect 250 μl of blood in an Eppendorf tube before sacrifice. Blood was left for 15 min at room temperature to promote clot formation before centrifugation (3500 g for 10 min at 4°C) and serum collection. Serum samples were stored at −80°C until analysis. CK activity was determined using the Creatine Kinase Activity Assay Kit (Sigma-Aldrich) and the Synergy 4 instrument (BioTek Instruments).
Egorova T.V., Zotova E.D., Reshetov D.A., Polikarpova A.V., Vassilieva S.G., Vlodavets D.V., Gavrilov A.A., Ulianov S.V., Buchman V.L, & Deykin A.V. (2019). CRISPR/Cas9-generated mouse model of Duchenne muscular dystrophy recapitulating a newly identified large 430 kb deletion in the human DMD gene. Disease Models & Mechanisms, 12(4), dmm037655.
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