Biotek synergy h1 plate reader

Manufactured by Agilent Technologies

Sourced in United States

The BioTek Synergy H1 plate reader is a multi-mode microplate reader designed for a variety of applications. It can measure absorbance, fluorescence, and luminescence in microplates. The Synergy H1 features a quadruple monochromator-based optical system and can accommodate a wide range of microplate formats.

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Lab products found in correlation

White 96 well plate, by PerkinElmer (1 mentions) Wallac 1420 victor plate reader, by PerkinElmer (1 mentions) Celltitre glo assay, by Promega (1 mentions) Biotek gen5, by Agilent Technologies (1 mentions) Synergy h1 plate reader, by Agilent Technologies (1 mentions) Dcf da, by Agilent Technologies (1 mentions) 96 well plate, by Corning (1 mentions) Microseal b pcr plate sealing film, by Bio-Rad (1 mentions) Celltiter glo assay, by Promega (1 mentions) Guanidine hcl, by Merck Group (1 mentions) Quant it picogreen, by Thermo Fisher Scientific (1 mentions) Celltiter glo, by Promega (1 mentions)

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12 protocols using biotek synergy h1 plate reader

Quantifying Intracellular ATP and Extracellular Lactate in CTB

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Isolated CTB were plated on flat-bottom 96-well plates as previously described and cultured in complete growth medium for 8 and 72 hours. The growth media was removed and 40 μL of the cell lysate was used to quantify intracellular ATP using an ATP luminescence assay (ATPlite^™, PerkinElmer^®). Luminescence was measured using a Biotek Synergy H1 plate reader (Biotek). ATP content was normalized to lysate protein concentration using a bicinchoninic acid assay assay (Thermo Pierce).
To measure extracellular lactate, medium was collected from CTB incubated in 96-well plates as described with fresh medium during an 8-hour period, representing lactate produced during 0–8 h or 64–72 h in culture. Extracellular lactate was measured using a colorometric lactate assay kit (Sigma) and plates were read using a Biotek Synergy H1 plate reader. Lactate measurements were normalized to DNA content using the Quant-iT™ PicoGreen^® dsDNA kit as outlined for metabolic flux analyses.

Kolahi K.S., Valent A.M, & Thornburg K.L. (2017). Cytotrophoblast, Not Syncytiotrophoblast, Dominates Glycolysis and Oxidative Phosphorylation in Human Term Placenta. Scientific Reports, 7, 42941.

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Stx Phage Induction and Mobilization

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All experiments were executed with two biological replicates. TT12A and TT12B strains were cultured overnight (o/n) at 37 °C with shaking (220 rpm) in LB medium. Bacterial o/n cultures were diluted to an OD₆₀₀ of 0.03 in fresh LB medium and grown at 37 °C with shaking (220 rpm) to early-log phase (OD₆₀₀~0.3) and then divided into two subcultures, LB and LB + Mitomycin C (MMC). Triggering the RecA-dependent SOS response with MMC constitutes a major pathway of Stx phage induction and mobilization [125 (link)]. Subculture LB + MMC was supplemented with MMC (Sigma-Aldrich, Saint Louis, MO, USA) at a final concentration of 0.5 μg/mL to mobilize carried prophages, while subculture LB was used to evaluate spontaneous prophage mobilization. Growth curves were recorded in a 96-well plate (Corning 3370, Corning Inc., Corning, NY, USA) at OD₆₀₀ on a BioTek Synergy H1 plate reader (BioTek Instruments, Inc., Winooski, VT, USA) for 16 hrs at 10 min intervals to assess prophage-induced bacterial lysis.

Kalalah A.A., Koenig S.S., Feng P., Bosilevac J.M., Bono J.L, & Eppinger M. (2024). Pathogenomes of Shiga Toxin Positive and Negative Escherichia coli O157:H7 Strains TT12A and TT12B: Comprehensive Phylogenomic Analysis Using Closed Genomes. Microorganisms, 12(4), 699.

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Evaluating Glioma Cell Viability

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Optimized number of U87MG, A172, U118, T98G and E98 cells (3 × 10³ to 5 × 10³) were seeded in white 96-well plates (Perkin Elmer) and allowed to attach. After 24 h, cells were treated with determined concentration of chemical compounds for 24–72 h. Cell viability was measured using a CellTitre Glo assay (Promega) according the manufacturer’s instructions. Luminescence was read with BioTek Synergy H1 plate reader (BioTek, Country). Bioluminescence was normalized and presented as a per cent of the control.
Cell viability for glioma stem cells was seeded into 96-well plates 5 × 10³ cells/well and allowed to attach overnight. Next day medium was changed into a medium containing drugs. After 72 h of treatment medium was changed into fresh NSC+/+ containing 10% of Alamar blue solution (10099022, Fisher Scientific). Fluorescent signal was measured after 210 min of incubation in 37°C using Wallac Victor 1420 plate reader (Perkin Elmer). Fluorescence was normalized and presented as a per cent of the control.

Merisaari J., Denisova O.V., Doroszko M., Le Joncour V., Johansson P., Leenders W.P., Kastrinsky D.B., Zaware N., Narla G., Laakkonen P., Nelander S., Ohlmeyer M, & Westermarck J. (2020). Monotherapy efficacy of blood–brain barrier permeable small molecule reactivators of protein phosphatase 2A in glioblastoma. Brain Communications, 2(1), fcaa002.

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Hepatocyte Oxidative Stress Assay

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Primary mouse hepatocytes were seeded on a 96-well black plate and, the following day, incubated with 100 nM of SD2267 for 6 h. Cells were then loaded with 50 μM of dichlorodihydrofluorescein diacetate (DCF-DA) (Cayman Chemical, MI, USA) in serum-free media for 30 min at 37 °C, washed with serum-free media, and treated with 20 mM of acetaminophen (APAP; Sigma-Aldrich, MO, USA). Fluorescence intensities (ex488/em525) were measured using a BioTek Synergy H1 plate Reader (BioTek) every 20 min for 10 h. AML12 cells were seeded on a 96-well black plate and, the following day, incubated with 5 mM of APAP in the absence or presence of SD2267. After 4 h, cells were stained with 50 μM of DCF-DA in serum-free media for 30 min at 37 °C, washed with serum-free media, and fluorescence intensities (ex488/em525) were measured using a BioTek Synergy H1 plate Reader. Data were analyzed using BioTek Gen5 software (BioTek).

Park S.Y., Gurung R., Hwang J.H., Kang J.H., Jung H.J., Zeb A., Hwang J.I., Park S.J., Maeng H.J., Shin D, & Oh S.H. (2023). Development of KEAP1-targeting PROTAC and its antioxidant properties: In vitro and in vivo. Redox Biology, 64, 102783.

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Cholic Acid Tolerance in E. faecalis

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A subset of transposon mutants that were underrepresented in the total E. faecalis Tn mutant population in the presence of cholic acid was selected for further analysis. Strains containing either the empty vector plasmid pMSP3535 or the appropriate pMSP3535 derivative were cultured to mid-logarithmic stage in MM9-YEG supplemented with Erm. Nisin was added for 1 h prior to starting growth curves to induce gene expression from the pMSP3535 constructs. A spectrophotometer was used to measure the OD₆₀₀ of each culture, and cultures were adjusted to an OD₆₀₀ of 0.05 in either MM9-YEG–Erm–nisin or MM9-YEG–Erm–nisin–0.15% cholic acid in a 96-well plate (Corning). Plates were sealed with Microseal B PCR plate sealing film (Bio-Rad). OD₆₀₀ values were measured at 15-min intervals for 10 h using a BioTek Synergy H1 plate reader (BioTek Instruments, Inc.). Experiments were repeated in triplicate (each with two technical duplicates), and error bars show the standard error of the mean. Statistical differences between endpoint OD₆₀₀ values were calculated using an unpaired Student t test with a significance cutoff of P < 0.05.

Dale J.L., Beckman K.B., Willett J.L., Nilson J.L., Palani N.P., Baller J.A., Hauge A., Gohl D.M., Erickson R., Manias D.A., Sadowsky M.J, & Dunny G.M. (2018). Comprehensive Functional Analysis of the Enterococcus faecalis Core Genome Using an Ordered, Sequence-Defined Collection of Insertional Mutations in Strain OG1RF. mSystems, 3(5), e00062-18.

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Canine Thymidine Kinase 1 ELISA Assay

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The Canine Thymidine Kinase 1 soluble ELISA assay (MyBiosource Inc, San Diego, CA) was used to evaluate TK1 levels in the dog followed longitudinally. The assay was performed according to the manufacturer’s protocol. Briefly, 40 µl of sample was added to wells followed by 10 µl anti-TK1 antibody. Then 50 µl streptavidin-HRP was added to each well except the blank control well. The plate was mixed well, covered with sealer and incubated for 60 min at 37 °C. The plate was then washed 5 times with wash buffer and the wells were soaked with at least 0.35 ml wash buffer for 30 s to 1 min for each wash. Next 50 µl of substrate solution A was added to each well followed by 50 µl of substrate solution B to each well. The plate was covered with a fresh sealer for 10 min at 37 °C in the dark. Finally, 50 µl of Stop Solution was added to each well. Plates were read at an absorbance of 450nm (BioTek Synergy H1 plate reader, BioTek Instruments, Winooski, VT) within 10 min of stop solution being added. The standard curve was linearized and fitted to a 5-parameter logistic curve using statistical software (Graphpad Software, version 8, San Diego, CA).

Wilson-Robles H., Miller T., Jarvis J., Terrell J., Kelly T.K., Bygott T, & Bougoussa M. (2021). Characterizing circulating nucleosomes in the plasma of dogs with hemangiosarcoma. BMC Veterinary Research, 17, 231.

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Measuring NADPH Oxidase Activity in Caco-2 Cells

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NADPH oxidase activity was measured using a lucigenin-enhanced chemiluminescence assay in membrane fractions from differentiated Caco-2 cells incubated in the absence or the presence of TNFα (5 ng/ml). For the isolation of membrane fractions, cells were homogenized in Krebs Buffer (20 mM HEPES, 119 mM NaCl, 4.7 mM KCl, 1 mM MgSO₄, 0.4 mM NaH₂PO₄, 0.15 mM Na₂HPO₄ and 1.25 mM CaCl₂) containing 1 mM PMSF, and Roche proteases inhibitor cocktail (Roche, Switzerland) and centrifuged at 800g for 10 min at 4 °C. The supernatant was subsequently centrifuged at 100,000g for 60 min at 4 °C, the pellet was collected (membrane fraction) and resuspended in Krebs buffer. Aliquots of membrane fractions (30 μg of protein) were added with or without 1 μM epicatechin or apocynin, and subsequently with 5 μM lucigenin and 50 μM NADPH. The reaction was followed under temperature-controlled conditions (37 °C). Light emission was measured every 30 s for 20 min using a Biotek Synergy H1 plate reader (BioTek Instruments, Inc.,Winooski, VT, USA) in the chemiluminescence mode. Results were expressed as the difference between the areas under the curve in the absence and in the presence of the NADPH oxidase inhibitor VAS-2780 (1 μM).

Cremonini E., Wang Z., Bettaieb A., Adamo A.M., Daveri E., Mills D.A., Kalanetra K.M., Haj F.G., Karakas S, & Oteiza P.I. (2017). (-)-Epicatechin protects the intestinal barrier from high fat diet-induced permeabilization: Implications for steatosis and insulin resistance. Redox Biology, 14, 588-599.

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Cell Viability Assay Protocol

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Optimized numbers of cells (2.5 × 10³ for T98G, U87MG and human fibroblasts or 5 × 10³ for E98, BT3-CD133⁺ and BT12) were plated onto 96-well plates and allowed to adhere. The next day, the cells were treated with vehicle (DMSO) or the indicated compounds. After 72 h, cell viability was measured using the CellTiter-Glo assay (Promega) according to the manufacturer’s instructions using a BioTek Synergy H1 plate reader (BioTek).

Denisova O.V., Merisaari J., Kaur A., Yetukuri L., Jumppanen M., von Schantz-Fant C., Ohlmeyer M., Wennerberg K., Aittokallio T., Taipale M, & Westermarck J. (2022). Development of actionable targets of multi-kinase inhibitors (AToMI) screening platform to dissect kinase targets of staurosporines in glioblastoma cells. Scientific Reports, 12, 13796.

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Fluorogenic ACE2 Activity Assay

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Renal and urinary ACE2 activity were measured using the specific fluorogenic ACE2 substrate, Mca-APK-(Dnp) (Biomol International, NY, United States) in the presence and absence of the ACE2 inhibitor, MLN-460 (gift from Millennium Pharmaceuticals, Cambridge, MA) as described before with some modifications (Chodavarapu et al., 2013 (link); Gutta et al., 2018 (link)). Diluted urine sample equivalent to 0.2 µg of creatinine and kidney lysates containing 1 µg of protein were pre incubated with 20 µl of assay buffer (50 mM Tris, 5 mM ZnCl₂, 150 mM NaCl₂ and 10 µM lisinopril) with or without MLN 4760 (10 μM) for 30 min at room temperature. After the incubation, 100 μL of the assay buffer containing the ACE2 substrate Mca-APK (Dnp) (50 μM) was added the reaction mixture and incubated for 15–60 min at room temperature. Florescence was measured at excitation (λ ex): 335 nm and emission (λ _em): 405 nm using Biotek Synergy H1 plate reader (BioTek instruments, Winooski, VT, United States).

Alawi L.F., Dhakal S., Emberesh S.E., Sawant H., Hosawi A., Thanekar U., Grobe N, & Elased K.M. (2021). Effects of Angiotensin II Type 1A Receptor on ACE2, Neprilysin and KIM-1 in Two Kidney One Clip (2K1C) Model of Renovascular Hypertension. Frontiers in Pharmacology, 11, 602985.

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Quantifying Cellular Staining Intensity

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SW1353 cells were seeded at a density of 4 × 10⁵ cells in 6 cm dishes. The cells were then treated with MIA and/or Se for 24 h. Following treatment, the culture medium was removed, and the cells were washed with phosphate-buffered saline (PBS). Subsequently, the cells were fixed using 4% paraformaldehyde in PBS for 10 min at room temperature (RT) and then rinsed with PBS. The fixed cells were then incubated with a 0.5% toluidine blue O staining solution for 30 min at RT. After washing with PBS, images of the staining were captured using a digital camera. To quantify the toluidine blue O pigment, it was extracted by incubating the cells with 6 mol/L guanidine-HCl (Sigma-Aldrich Co., LLC, St. Louis, MO, USA) for 10 min at RT. The absorbance of the extracts was measured at 630 nm using a microplate reader (BioTek Synergy H1 Plate Reader, BioTek Instruments Inc., Winooski, VT, USA). This procedure was performed to assess and quantify the staining intensity. Each experiment was carried out in triplicate for accuracy and consistency.

Cheng H.L., Yen C.C., Huang L.W., Hu Y.C., Huang T.C., Hsieh B.S, & Chang K.L. (2024). Selenium Lessens Osteoarthritis by Protecting Articular Chondrocytes from Oxidative Damage through Nrf2 and NF-κB Pathways. International Journal of Molecular Sciences, 25(5), 2511.

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