Synergy h1 reader

Manufactured by Agilent Technologies

Sourced in United States

The Synergy H1 reader is a multi-mode microplate reader designed for a wide range of applications. It features a high-performance monochromator-based optical system and supports absorbance, fluorescence, and luminescence detection modes.

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Lab products found in correlation

Gen5 2, by Agilent Technologies (3 mentions) Turbo dna free kit, by Thermo Fisher Scientific (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Gsh gssg assay kit, by Nanjing Jiancheng (1 mentions) Black 96 well plate, by Corning (1 mentions) Bactiter glo microbial cell viability assay solution, by Promega (1 mentions) Biolux gaussia luciferase assay kit, by New England Biolabs (1 mentions) Nano glo luciferase assay system, by Promega (1 mentions) Mitocheck complex 1 activity assay kit, by Cayman Chemical (1 mentions) Pmv306hsp luxg13, by Addgene (1 mentions) Middlebrook 7h9 medium, by BD (1 mentions) Folin ciocalteu reagent, by Merck Group (1 mentions) Deproteinization sample preparation kit, by Abcam (1 mentions) Atp colorimetric fluorometric assay kit, by Abcam (1 mentions) Catalase assay kit, by Nanjing Jiancheng (1 mentions) Cell counting kit 8 reagent, by AbMole (1 mentions) Celltiter glo atp based assay, by Promega (1 mentions) 96 well clear flat bottom tc treated culture microplate, by BD (1 mentions) H2dcfda, by Thermo Fisher Scientific (1 mentions) Pyruvate colorimetric fluorometric assay kit, by Abcam (1 mentions)

58 protocols using synergy h1 reader

Cell Cytotoxicity and Caspase-3 Assay

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The effect of NMN on cell cytotoxicity was determined using the Spectrophotometric LDH Determination Kit (Solarbio, Beijing, China). The cells were added 1 mL LDH extract to be sonicated (ultrasound for 3 s, 10‐s interval, repeated 30 times) and then centrifuged at 8000 g at 4 °C for 10 min, and the supernatant was measured on Synergy H1 Reader (BioTek) at 450nm absorbance. The result was expressed as U·mg⁻¹ protein. In addition, Colorimetric Caspase‐3 Activity Assay Kit (Solarbio) was used to determine caspase‐3 activity. The cells were lysed by shaking with 200 µL caspase‐3 lysis solution, ice‐bathed for 10 min, shaken again and then centrifuged at 12 000 g at 4 °C for 10 min, and the supernatant was taken to determine the OD value at 405nm using Synergy H1 Reader (BioTek). The percentage increase in caspase activity was expressed by the following formula: 100%×[(experimental treatment group OD ‐ blank control OD)/(experimental control OD ‐ blank control OD)].

Deng X., Liang X., Yang H., Huang Z., Huang X., Liang C., Kuang Y., Qin Y., Lin F, & Luo Z. (2021). Nicotinamide mononucleotide (NMN) protects bEnd.3 cells against H2O2‐induced damage via NAMPT and the NF‐κB p65 signalling pathway. FEBS Open Bio, 11(3), 866-879.

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RNA Extraction from Rhizobium leguminosarum

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Cultures of R. leguminosarum bv. trifolii TA1 and ΔpssA mutant strains were grown in triplicate for 24 h in 79CA at 28 °C with shaking, then diluted to an OD₆₀₀ of 0.05 in fresh 79CA medium and incubated until an OD₆₀₀ of 0.7 was reached (≈10⁹ CFU). The cells were harvested by centrifugation at 4 °C and immediately submitted for RNA extraction with the commercial GeneMATRIX Universal RNA Purification Kit (EURx Sp. z o.o., Gdańsk, Poland) according to the manufacturers protocol. Contaminating DNA was removed with TURBO DNA-free™ Kit (Thermo Fisher Scientific, Waltham, MA, USA), under rigorous treatment protocol, with minor modifications. The quantity and quality of RNA were checked spectrophotometrically (Synergy H1 reader, Agilent Technologies, Inc., Santa Clara, CA, USA), fluorometrically (Qubit 2.0 Fluorometer with Qubit RNA High Sensitivity (HS) Assay Kit, Thermo Fisher Scientific, Waltham, MA, USA), in microcapillary electrophoresis (2100 Bioanalyzer Instrument with RNA 6000 Nano Kit, Agilent Technologies, Inc., Santa Clara, CA, USA), and in the PCR reaction. Details regarding the optimization of high-quality RNA extraction were presented in Supplementary File S1.

Marczak M., Żebracki K., Koper P., Horbowicz A., Wójcik M, & Mazur A. (2023). A New Face of the Old Gene: Deletion of the PssA, Encoding Monotopic Inner Membrane Phosphoglycosyl Transferase in Rhizobium leguminosarum, Leads to Diverse Phenotypes That Could Be Attributable to Downstream Effects of the Lack of Exopolysaccharide. International Journal of Molecular Sciences, 24(2), 1035.

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Quantifying Cochlear MDA Levels

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The measurement of malondialdehyde (MDA) was conducted using the MDA assay kit (Biosharp, Beijing, China, BL904A) following the manufacturer's instructions. This assay is based on the reaction of MDA with thiobarbituric acid (TBA), resulting in the formation of a highly absorbent adduct that can be easily quantified using a spectrophotometer at 532 nm. To perform the assay, 25 cochlear explants were collected and homogenized with an ultrasonic homogenizer in 120 μL of PBS on ice. Subsequently, 100 μL of the supernatant was utilized for the MDA analysis using a BioTek Synergy H1 reader (Agilent, Santa Clara, CA, U.S.). The MDA concentration is expressed in nanomoles (nM) per 10 mg of tissue.

Li Y., Wu J., Yu H., Lu X, & Ni Y. (2023). Formononetin ameliorates cisplatin-induced hair cell death via activation of the PI3K/AKT-Nrf2 signaling pathway. Heliyon, 10(1), e23750.

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Glutathione Redox Assay in Cochlear Explants

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The GSH/GSSG assay was conducted using the GSH/GSSG assay kit (Jiancheng, Nanjing, China, A061-1-1) following the manufacturer's instructions. This method is based on the reaction between GSH and 5,5′-di-thiobis (DTNB), which leads to the cyclic production of TNB. The constant production of TNB is then measured using spectrophotometry at a wavelength of 405 nm. To perform the assay, a total of twenty-five cochlear explants were collected and homogenized using an ultrasonic homogenizer in 120 μL of the provided homogenization reagent on ice. The concentrations of total glutathione (tGSH) and oxidized glutathione (GSSG) were determined using a BioTek Synergy H1 reader (Agilent, Santa Clara, CA, U.S.). The levels of reduced glutathione (GSH) were calculated as follows: GSH = tGSH - (GSSG * 2). The concentration of GSSG is presented as nanomoles per 10 mg of tissue.

Li Y., Wu J., Yu H., Lu X, & Ni Y. (2023). Formononetin ameliorates cisplatin-induced hair cell death via activation of the PI3K/AKT-Nrf2 signaling pathway. Heliyon, 10(1), e23750.

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Caspase-11 Inhibitor Screening Assay

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Caspase-11 (9 U/μL in 10 μL caspase activity buffer (100 mM 2-(N-morpholino)ethanesulfonic acid (MES)), pH 6.5, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 10% poly(ethylene-glycol) (PEG)-8000, 10 mM dithiothreitol (DTT)) was added to 10 μL of caspase-11 inhibitor (5 μM), THPI (5 μM), or no inhibitor in caspase activity buffer. For testing THPIs as inhibitors, enzyme was incubated with THPIs for 2 h at 37 °C. Residual enzyme activity was monitored by adding 10 μL of caspase substrate in caspase activity buffer to produce a final concentration of <0.1K_M. pNA cleavage from the peptide was monitored at 37 °C over 1 h using λ = 405 nm on a Bio-Tek Synergy H1 Reader or H4 Hybrid Reader.

Knapinska A.M., Hart M., Drotleff G, & Fields G.B. (2019). Matrix Metalloproteinase Triple-Helical Peptide Inhibitors: Potential Cross-Reactivity with Caspase-11. Molecules, 24(23), 4355.

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Intracellular ATP Quantification Protocol

Cited 1 time

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Intracellular ATP was estimated as described (33 (link)). Briefly, 150 μl of cell cultures were aliquoted into PCR tubes, immediately heated at 80° C for 10 min, and subsequently placed on ice. At the same time, 1 ml of each of these cultures was transferred to a 1.5 ml tube and placed on ice. Using a multichannel pipette, 30–50 μl of heat-inactivated (80° C for 10 min) cells were transferred to 96-well black plates (Corning) containing 150 μl of BacTiter-Glo Microbial Cell Viability Assay Solution (Promega). Samples were mixed by pipetting, and their luminescence was measured in a Synergy H1 Reader (BioTek). Cells from the 1 ml of culture aliquots were collected by centrifugation (4° C at 14,000 rpm for 5 min), resuspended in 250 μl of 0.5% SDS, and lysed by boiling at 100° C for 10 min. The concentrations of proteins in the cell lysates were estimated using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein concentrations in these samples were used to calculate the protein amount present in the luminescence reactions. ATP levels were calculated by normalizing luminescence values of each sample by their protein content.

Pontes M.H, & Groisman E.A. (2019). Slow growth dictates non-heritable antibiotic resistance in Salmonella enterica. Science signaling, 12(592), eaax3938.

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Luciferase Activity Quantification

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Gaussia luciferase activity was measured with the BioLux Gaussia luciferase assay kit (New England BioLabs). A 50-μl volume of cleared culture supernatant was added into a 96-well white plate, and 20 μl of GLuc assay solution was prepared and thoroughly mixed with the sample immediately before luminescence detection with a Synergy H1 reader (Biotek). NanoLuc activity was measured with Nano-Glo luciferase assay system (Promega). A 25-μl volume of cleared culture supernatant was added into a 96-well white plate, and 25 μl of NanoLuc assay solution was prepared and thoroughly mixed with the sample before luminescence detection with a Synergy H1 reader.

Gan T., Zhou D., Huang Y., Xiao S., Ma Z., Hu X., Tong Y., Yan H, & Zhong J. (2021). Development of a New Reverse Genetics System for Ebola Virus. mSphere, 6(3), e00235-21.

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Anti-CRISPR Assay for Promoter Activity

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DNA encoding a crRNA targeting the constitutive promoter region of J23119 was cloned into the BsaI site of the pCRISPathBrick plasmid (Cress et al., 2015 (link)). This plasmid was co-transformed into E. coli BL21 cells with pCM-str, a plasmid in which the J23119 artificial promoter drives constitutive expression of the luxCDABE operon from Photorhabdus luminescens (Winson et al., 1998 (link)). These cells were then co-transformed with a pCDF-1b plasmid expressing the anti-CRISPR proteins and a protospacer targeting the J23119 promoter. Cells containing the three plasmids were grown in LB supplemented with kanamycin, chloramphenicol and streptomycin until they reached an optical density at 600 nm (OD₆₀₀) of 0.6. The cultures were then diluted to an OD₆₀₀ of 0.1 in LB containing 200 ng/mL aTc, 0.2% arabinose and 0.01 mM IPTG, and 100 μl was dispensed into a 96-well plate. The plate was incubated with shaking at 37°C using a Synergy H1 reader controlled by Gen5 2.09 software (BioTek Instruments Inc.), and the OD₆₀₀ and luminescence was monitored for 24 hours.

Garcia B., Lee J., Edraki A., Hidalgo-Reyes Y., Erwood S., Mir A., Trost C.N., Seroussi U., Stanley S.Y., Cohn R.D., Claycomb J.M., Sontheimer E.J., Maxwell K.L, & Davidson A.R. (2019). Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing. Cell reports, 29(7), 1739-1746.e5.

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Quantifying Cellular NAD+/NADH Levels

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The NAD⁺ and NADH content of silver ion medium treated-cells was measured using a colorimetric NAD⁺/NADH ratio assay kit (AAT Bioquest, Sunnyvale, CA, USA) according to the manufacturer’s instruction. Briefly, cells were collected and resuspended with lysis buffer for 15 minutes. The lysate was centrifuged at 1500 rpm for 5 minutes and the supernatant was used for tests. The pool of NAD⁺ and NADH and the NAD⁺ extract were incubated with reaction mixture at room temperature, simultaneously. Three hours later, the absorbance at 460 nm was monitored by Synergy H1 Reader (BioTek, Winooski, VT). The amount of absorbance signal is directly proportional to the concentration of NAD⁺ and NADH, which is used as an indicator of the cellular NAD⁺ and NADH concentration. Experiments were repeated four times.

Zhao Y., Bunch T.D, & Isom S.C. (2021). Effects of electrical biostimulation and silver ions on porcine fibroblast cells. PLoS ONE, 16(2), e0246847.

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Spectrophotometric Assay of Complex I

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Determination of the activity of complex I was performed using a MitoCheck® Complex I Activity Assay Kit (Cayman Chemical, Ann Arbor, MI, USA) in accordance with the manufacturer’s instructions. Absorbance at 340 nm was measured using a Synergy H1 Reader (BioTek). The assay was performed in kinetic read mode at 25 °C and absorbance was monitored every 30 s for 15 min. Complex I activity was determined as a ratio of the rate of sample well and the rate of vehicle control.

Liskova V., Kajsik M., Chovancova B., Roller L, & Krizanova O. (2022). Camptothecin, triptolide, and apoptosis inducer kit have differential effects on mitochondria in colorectal carcinoma cells. FEBS Open Bio, 12(5), 913-924.

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