Synergy htx multi mode reader

Manufactured by Agilent Technologies

Sourced in United States, Germany, Belgium, United Kingdom, Switzerland, China

The Synergy HTX Multi-Mode Reader is a versatile instrument designed for various laboratory applications. It features a compact design and supports multiple detection modes, enabling researchers to perform diverse assays and analyses.

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387 protocols using synergy htx multi mode reader

Protein and Growth Factor Release Kinetics

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Aliquots of the multifunctional MeLam microparticles were suspended in 5 mL PBS, samples were gently shaked at 60 rpm in a water bath at 37°C. At defined time intervals, 500 μL of PBS were removed and replaced with 500 μL of fresh PBS. The removed supernatants were stored frozen until required and were then assayed for total protein content using the Micro BCA assay kit. Briefly, 50 μL of the collected samples were diluted in 100 μL of PBS, mixed with 150 μL of the Micro BCA working solution and incubated for 2 hours at 37°C. Afterwards, the quantity of protein was measured by the absorbance at 592 nm in a microplate reader (Synergy HTX multi-mode reader, Biotek Instruments, Inc, USA). The protein release profile was calculated following equation (2) (Che et al., 2015 ):

C u m u l a t i v e P L r e l e a s e (%) = \frac{V_{e} \sum_{i}^{n - 1} C_{i} + V_{0} C_{n}}{m_{P L}} \times 100

Where is V_e = 500 μL; V₀= 5 mL; C_i is the concentration of total protein released from MeLam microparticles at the time and m_PL is the weight of PL used for the release.
ELISA assay (ThermoFisherScientific, USA) was also performed to evaluate the release of transforming growth factor (TGF-β1) and vascular epithelial growth factor (VEGF) from the MeLam microparticles. The assay was performed according to the manufacturer’s standard protocols. The optical density values were measured using a Synergy HTX multi-mode reader (Biotek Instruments, Inc, USA) set at 450 nm.

Martins C., Custódio C, & Mano J. (2018). Multifunctional laminarin microparticles for cell adhesion and expansion. Carbohydrate polymers, 202, 91-98.

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Mitochondrial PDH Activity Assay

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The PDH activity was measured using the PDH activity colorimetric assay kit (BioVision, Milpitas, CA, USA). Briefly, 20 μg of isolated mitochondria from gastrocnemius muscle were resuspended in 50 μl PDH assay buffer, then 50 μl reaction mix (containing 46 μl PDH assay buffer, 2 μl PDH developer, and 2 μl PDH substrate) was added. The absorbance at 450 nm was measured immediately using Synergy HTX Multi-Mode Reader (BioTek, Winooski, VT, USA) in the kinetic mode for 60 min at 37°C.

Zhang R., Hou T., Cheng H, & Wang X. (2019). NDUFAB1 protects against obesity and insulin resistance by enhancing mitochondrial metabolism. The FASEB Journal, 33(12), 13310-13322.

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Proliferative Capability of BON1 Cells

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The proliferation capability of BON1 cells was firstly assessed by a MTS cell proliferation colorimetric assay kit (Biovision, Milpitas, CA, USA) according to the manufactures instructions. Briefly, BON1 cells (5 × 10³/well) were seeded in 96-well plate and incubated for 3 days with IL-6 (25 ng/mL, Cell Signaling) or CRP (20 µg/mL, R&D Systems). Twenty microliters of MTS reagent were added to each well. After 3 h of incubation, optical density was read at 490 nm and 650 nm using a SYNERGY/HTX multi-mode reader (BioTek Instruments).
Secondly, we used the BrdU Cell Proliferation ELISA Kit (Abcam), in order to assess the same mechanism, according to the manufactures instructions.

Schimmack S., Yang Y., Felix K., Herbst M., Li Y., Schenk M., Bergmann F., Hackert T, & Strobel O. (2019). C-reactive protein (CRP) promotes malignant properties in pancreatic neuroendocrine neoplasms. Endocrine Connections, 8(7), 1007-1019.

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Dual Luciferase Assay for circRNA-miRNA Interaction

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The dual luciferase assay kit (cat. no. KGAF040) was purchased from KeyGEN BioTECH (Nanjing, Jiangsu, China), and all the plasmids were constructed by Sangon Biotech Company (Shanghai, China). Reporter assays were performed using the dual luciferase assay system as described previously [36 (link), 37 (link)]. The luciferase reporter constructs were generated from the miRNA target expression vector pmirGLO (Promega, Madison, WI, USA). Full-length binding-site-mutant and wild-type circRNA_37492 constructs, truncated mutant and wild-type 3’UTR of fibrinogen beta chain (Fgb) gene were individually co-transfected with miR-7682-3p mimics or Control into BUMPT cells for 48 h. Luciferase activities were then measured using a Synergy™ HTX multi-mode reader (Biotek, Winooski, VT, USA) and normalized according to renilla luciferase activity.

Cheng X., Ai K., Yi L., Liu W., Li Y., Wang Y, & Zhang D. (2022). The mmu_circRNA_37492/hsa_circ_0012138 function as potential ceRNA to attenuate obstructive renal fibrosis. Cell Death & Disease, 13(3), 207.

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Catalase Activity Measurement Protocol

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Catalase (CAT) activity was measured following the adapted protocol first described by Beers and Sizer [33 (link)]. Calibration was performed using three mL of Potassium phosphate buffer (K₃PO₄; Sigma-Aldrich, Schnelldorf, Germany). Samples (100 µL) and 2.9 mL of hydrogen peroxide (0.036 mol L⁻¹ H₂O₂; Sigma-Aldrich, Schnelldorf, Germany) were added to 96-well quartz microplates. Absorbance was read at 240 nm every 42 s over four minutes with a microplate reader (Biotek Synergy™ HTX Multi-Mode Reader, Winooski, VT, USA). Catalase activity was calculated using the H₂O₂ molar extinction coefficient (0.0436 ε^{mmol L−1}). Results were expressed relative to the total protein content (nmol min⁻¹ mg⁻¹ total protein).

Varela J., Martins S., Court M., Santos C.P., Paula J.R., Ferreira I.J., Diniz M., Repolho T, & Rosa R. (2023). Impacts of Deoxygenation and Hypoxia on Shark Embryos Anti-Predator Behavior and Oxidative Stress. Biology, 12(4), 577.

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Ubiquitin Quantification by ELISA

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Ubiquitin (Ub) content was determined through an ELISA as described by Lopes et al. [37 (link)]. Each sample (100 μL) was added to 96-well microplates (Greiner, Bio-One, Kremsmünster, Austria) and incubated overnight at 4 °C. After 24 h, microplates were washed two times with PBS containing 0.05% TWEEN 20, and 100 μL of blocking solution (1% BSA) was added to each well. The microplates were then incubated for 90 min at 37 °C. Subsequently, 50 μL of primary antibody (P4D1, sc-8017, HRP conjugated; Santa Cruz, CA, USA) was added to each well. After another overnight incubation period at 4 °C, microplates were washed three times to remove non-linked antibodies. Afterwards, 100 μl of substrate (TMB/E, Merck Millipore, St. Louis, MO, USA) was added to each microplate well and let to incubate for about 30 min at room temperature. Then 100 μL of stop solution (1 mol L⁻¹; HCl; Panreac, Barcelona, Spain) was added to each well. Absorbances were read at 450 nm, using a microplate reader (Biotek Synergy™ HTX Multi-Mode Reader, Winooski, VT, USA). The Ub content was calculated from the calibration curve, based on serial dilutions of purified ubiquitin (0–1 μg mL⁻¹, UbpBio, E-1100, Dallas, TX, USA). Results were expressed as µg mg⁻¹ total protein.

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Measuring EV Protein Content via MicroBCA

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The total EV protein content was measured using a MicroBCA protein assay. EV samples were diluted in 1x RIPA lysis buffer containing aprotinin (10 μg/mL) and were kept on ice for 15 min to lyse the EVs. A 150 μL volume of each sample was added to an equal volume of the MicroBCA working reagent in a 96 well-plate and incubated for 2 h at 37 °C per the manufacturer’s instructions. Protein concentration was quantified by measuring the absorbance at 562 nm on a microplate reader (Synergy HTX multimode reader, Bio-Tek Instruments Inc.).

D’Souza A., Burch A., Dave K.M., Sreeram A., Reynolds M.J., Dobbins D.X., Kamte Y.S., Zhao W., Sabatelle C., Joy G.M., Soman V., Chandran U.R., Shiva S.S., Quillinan N., Herson P.S, & Manickam D.S. (2021). Microvesicles Transfer Mitochondria and Increase Mitochondrial Function in Brain Endothelial Cells. Journal of controlled release : official journal of the Controlled Release Society, 338, 505-526.

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ATP Modulation in Ischemic Injury

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ATP5A doses of 100, 200, and 300 ng per 0.32 cm² of 96-well plate were used in these studies. The EV-ATP5A complexes were diluted in OGD media before addition to cells 4 h post-OGD exposure. The cells were incubated with the indicated samples for 4 h, washed with pre-warmed PBS, and resulting ATP levels were determined by CellTiter Glo assay (as described in section 2.11.2). The effects of the treatment were expressed as the resulting ATP levels compared to the untreated OGD cells subjected to reoxygenated/normoxic conditions for 4 h. Relative luminescent signals were measured using Synergy HTX multimode reader (Bio-Tek Instruments Inc., USA) at 1 sec integration time. The relative ATP levels (%) was calculated after normalizing the relative luminescence units (RLU) of treated cells to those untreated cells as shown in equation 3.

ATP levels (%) = \frac{RLU from treated cells}{RLU from untreated OGD cells} * 100

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Cytotoxic Effects of Bisporitin on Keratinocytes

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The cytotoxic effect of bisporitin was evaluated on HaCaT and A431 cell lines, respectively, human normal and tumoral keratinocytes, as well as HeLa cells from cervical carcinoma, by performing the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide reduction inhibition assay (MTT assay) [34 (link)], designed to be used for the spectrophotometric quantification of cell metabolic activity. Briefly, 3 × 10³ cells were seeded into a 96-well plate and incubated at 37 °C in the presence of 5% CO₂. The medium was then replaced with 100 μL of fresh medium containing bisporitin solution at a final concentration ranging from 0 to 10 μM/well. After 24, 48, and 72 h of incubation at 37 °C, the medium was removed, and 100 μL of 0.5 mg/mL MTT solution diluted in Dulbecco’s modified Eagle’s medium (DMEM) purchased from Lonza (Basel, Switzerland) without red phenol was added. After 4 h of incubation at 37 °C, the resulting insoluble formazan salts were solubilized in anhydrous isopropanol containing 40 mM HCl and quantified by measuring the absorbance at λ = 570 nm, using an automatic plate reader spectrophotometer (Synergy HTX Multi-Mode Reader-BIOTEK, Winooski, VT, USA). Cell survival was expressed as mean of the percentage values compared to control represented by untreated cells.

Ragucci S., Hussain H.Z., Bosso A., Landi N., Clemente A., Pedone P.V., Pizzo E, & Di Maro A. (2023). Isolation, Characterization, and Biocompatibility of Bisporitin, a Ribotoxin-like Protein from White Button Mushroom (Agaricus bisporus). Biomolecules, 13(2), 237.

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Human VEGF ELISA Sandwich Assay

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Human VEGF sandwich assay ELISA (Enzyme-linked Immunosorbent Assay) (Sigma-Aldrich) was performed based on supplier’s instructions. Briefly, 100 μl of standard or sample was pipetted to each well that was layered with capture antibody and placed in an incubator for 2.5 hours. Successively 100 μl of detection antibody was added to each well and incubated for 1 hour; 100 μl of streptavidin for 45 min; 100 μl of substrate solution for 20 min and finally 50 μl of stop solution. Each step was followed by four washing cycles with wash buffer solution. Light of 450 nm wavelength was employed to measure the assay using a plate reader (BIOTEK Flourescent plate reader, Synergy HTX multimode reader).

Bandaru P., Cefaloni G., Vajhadin F., Lee K., Kim H.J., Cho H.J., Hartel M.C., Zhang S., Sun W., Goudie M.J., Ahadian S., Dokmeci M.R., Lee J, & Khademhosseini A. (2020). Mechanical cues regulating proangiogenic potential of human mesenchymal stem cells through YAP-mediated mechanosensing. Small (Weinheim an der Bergstrasse, Germany), 16(25), e2001837.

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