Intracellular GSH content in treated and untreated cells was measured using the Glutathione Fluorometric Assay Kit (Biovision, USA) according to the manufacturer's instructions. Briefly, the sample lysate was mixed with the reaction buffer provided in the kit and the concentration of GSH was calculated from the GSH standard curve generated using the readings on Synergy H1 Hybrid reader (BioTek). For the estimation of lipid peroxidation products, the treated and untreated cells were washed twice with PBS and suspended in 1 ml of 15% sodium dodecyl sulfate (SDS)-PBS solution. The fluorescence intensities of the total fluorescent lipid peroxidation products was then obtained [13 (link)] through readings on Synergy H1 Hybrid reader (BioTek) with excitation at 360 nm and emission at 430 nm.
Synergy h1 hybrid reader
Manufactured by Agilent Technologies
Sourced in United States, Germany, Switzerland, Canada, United Kingdom
The Synergy H1 Hybrid Reader is a compact and versatile instrument designed for a wide range of microplate-based assays. It combines multiple detection modes, including absorbance, fluorescence, and luminescence, to provide a comprehensive analysis platform for life science research and drug discovery applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (10 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (5 mentions)
Sytox green, by Thermo Fisher Scientific (5 mentions)
Dual luciferase assay kit, by Promega (4 mentions)
Gene5, by Agilent Technologies (4 mentions)
Xylocaine, by Pfizer (4 mentions)
Luciferase assay system, by Promega (4 mentions)
Prism 7, by GraphPad (4 mentions)
96 well plate, by Corning (3 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (3 mentions)
Mtt dye, by Merck Group (3 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (3 mentions)
Xm10 monochromatic ccd camera, by Olympus (3 mentions)
Inverted microscope, by Olympus (3 mentions)
Gen5 data analysis software, by Agilent Technologies (3 mentions)
Crystal violet, by Merck Group (3 mentions)
Ix50 inverted fluorescence microscope, by Olympus (3 mentions)
Cellsens dimension software, by Olympus (3 mentions)
Reporter lysis buffer, by Promega (3 mentions)
Calcein am, by Thermo Fisher Scientific (2 mentions)
445 protocols using synergy h1 hybrid reader
1
Intracellular GSH and Lipid Peroxidation Assay
2
Cytotoxicity Evaluation of Herbal Extracts
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Cell proliferation and IC50 were measured by MTT assay using multiple doses of the crude extracts, 4 A and the nine TLC fractions, on the aforementioned cell lines. The MTT salt is cleaved by dehydrogenase in the living cells and reduced to an insoluble formazan crystal which shows a specific purple color. The color was detected by a plate reader (Synergy H1 Hybrid Reader Biotek). Caski and HFL1 cell lines were seeded overnight in 96-well plate at an 8 × 103 cells/well density. After incubation, cells were treated with corresponding compounds dose and incubated for another 24 hours. Cell suspensions were removed, added 150 μl of MTT solution and incubated further for 1 hour. The solution was removed and added 100 μl DMSO added to dissolve the formazan product and incubated for about 5–10 minutes in a plate shaker. After the incubation, the absorbance was measured at 570 nm in a plate reader (Synergy H1 Hybrid Reader Biotek).
3
ELISA Analysis of SspA-1, SspA-2 Interaction with C3a, C5a
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ELISA was used to analyse the interaction of SspA-1, SspA-2 with C3a, C5a protein as described previously [20 (link)]. For C3a and C5a bound assay, 0–7 μg/mL SspA-1T, SspA-2T and BSA as a negative control were immobilized to 96-well plate (Corning, USA), 5 μg/mL C3a and C5a were applied after blocking with 1% BSA in PBST (PBS with 20% Tween-20), respectively. Mouse C3a/C3a des Arg monoclonal antibody (Abcam, England) or rabbit C5a monoclonal antibody (Abcam) was applied at 37 °C for 1 hour to test the direct binding capacity. For competitive binding assay, 5 μg/mL SspA-1T, SspA-2T and BSA were immobilized to 96-well plate, 5 μg/mL C5a combined with 0–20 μg/mL C3a were applied after blocking with 1% BSA in PBST, respectively. Mouse anti-C5 monoclonal antibody (Proteintech, Wuhan, China) was applied at 37 °C for 1 hour to test the C5a binding capacity. The HRP-conjugate goat-anti-rabbit IgG (CST) or HRP-conjugate rabbit-anti-mouse IgG (CST) was used as the second antibody at 37 °C for 1 h before Chromogenic reaction by TMB Chromogenic Reagent kit (Sangon) and detected by SynergyTM H1 Hybrid Reader (Biotek, USA). These data were presented as means ± standard deviations (SD) from three replicates. The experiments were repeated for three times.
4
Quantifying COX-Mediated PGE2 Synthesis
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Inhibition of COX activity was determined by measuring the synthesis of PGE2 according to the instructions provided with the kit. Brief, COX-1 or COX-2 enzyme and heme were added to test tubes containing COX reaction buffer (total of 0.5 mL). The mixture was vortex mixed and exposed to vehicle (DPBS) or CAPE (10 mg/mL), quercetin or myricetin (10 μM) for 10 min at 37°C. This was followed by the addition of AA (100 μM) with further incubation for 2 min. Hydrochloric acid (1 M) was added to stop the COX reaction followed by chemical reduction with stannous chloride solution. A standard curve with PGE2 was generated at the same time and from the same plate and was used to quantify PGE2 levels produced in the presence of test compounds. Absorbance was read using a plate reader (SynergyTM H1 Hybrid Reader, Biotek, United States) at 412 nm. Diclofenac (10 μM) was used as the reference compound, and all compounds were diluted in a buffer kit.
5
Evaluating Platelet Viability with MTT Assay
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The MTT assay was performed according to Mosmann (1983) (link). Using 96-well plates, platelets (1.5 × 108 platelets/mL) were incubated with CAPE (0.25, 0.5, 1, 2.5, 5, and 10 mg/mL) or quercetin or myricetin (10 μM) for 5, 15, 30, and 60 min. After the incubation period, platelets were incubated with 5 mg/mL of MTT solution for 3 h in a CO2 incubator. Then MTT dye was removed and 100 μL of solubilization solution (SDS 10% acidified) were added to the wells. Absorbance was measured at 540 nm using a microplate reader (SynergyTM H1 Hybrid Reader, BioTek, United States). The positive control was 10% triton-X.
6
Quantification of Methylglyoxal and AGEs in Serum
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Peripheral blood (0.5 ml) was collected by the intracardiac puncture. Blood was centrifuged at 5,000 × g for 10 min at 4°C and serum was transferred to microcentrifuge tubes. To measure MGO levels in serum, the samples were deproteinized using Deproteinizing Sample Preparation Kit - TCA (Abcam ab204708) according to the manufacturer’s instructions. Serum levels of MGO were measured using ELISA competitive kit for OxiSelect™ Methylglyoxal (Catalog No. STA-811, Cell Biolabs, San Diego, CA, United States). F-AGEs in serum were measured diluting the samples 1:2 in phosphate-buffered saline (PBS, pH 7.4), and transferred to black 96-well plates, 200 µl total per well. Fluorescence spectra were recorded in duplicate on a fluorometer (SynergyTM H1 Hybrid Reader, Biotek, United States). The excitation and emission wavelengths were 360 and 447 nm, respectively, and the PBS solution was used as blank. Individual concentrations were normalized by blank fluorescence. The fluorescence intensity was expressed in arbitrary units (a.u.). To assess glucose concentration, animals fasted for 8 h, blood from the tail vein was collected and glucose was measured using ACCUCHECK Blood Glucose (Monitoring System®, Roche Diagnostics, Indianapolis, United States).
7
Chlorogenic Acid Endothelial Cell Viability
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Due to the short platelet half-life of, an endothelial cell line was employed for cytotoxicity studies. The effect of chlorogenic acid on endothelial cell viability was assessed using a Vybrant MTT Cell Proliferation Assay Kit (Invitrogen). Briefly, confluent cells were seeded in 96-well plates after a 4-hour chlorogenic acid treatment (0.05 to 1 mmol/L) and incubated for 3 hours with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 12 mmol/L [23] (link). Then, SDS-HCl solution was added to each well and after 16 hours, the absorbance was measured at 570 nm (BioTeK Synergy H1 Hybrid Reader). Cells treated with medium only were used as a negative control group. Cell viability was expressed as a percentage relative to the untreated control cells.
8
Luciferase Reporter Assay for FMR1 3'UTR
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The FMR1 3′-UTR target site sequence and sequence with an eight-nucleotide mutation in the miR-323a-3p target site were synthesized and cloned downstream of the luciferase gene in the pEGFP-C1 luciferase vector (Generay, China). The resulting vectors were named FMR1-3′-UTR-WT and FMR1-3′-UTR-Mut, respectively. Cells were seeded into 12-well plates and co-transfected with the constructed plasmids, internal control Renilla luciferase plasmid (pRL-SV40), and mimic or mimic NC using Lipofectamine 2000 (Invitrogen, USA). After 48 h, cells were harvested and luciferase activity measured using a Dual Luciferase Assay Kit (Promega, USA) and a Synergy H1 hybrid reader (Biotek, USA). Results were normalized to Renilla luciferase activity and the data expressed as relative luciferase activity. Experiments were performed in triplicate on three separate occasions.
9
Tracking Nanomaterial Biodistribution in Mice
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To collect urine and feces individually from each mouse, mice were placed in metabolic cages for a single mouse (Tecniplast) immediately after nanomaterial injection in accordance with the conditions for animal care recommended by the IACUC. The urine samples were collected after each spontaneous urination for the first 10 hrs and then after 24 hrs (just before euthanasia). Fluorescence of separate samples was measured immediately after their collection. Next, all urine samples from one animal were pooled, the volume was recorded and fluorescence of 100 µL of this 24-hrs urine sample was analyzed. Similarly, feces were collected every 1 hr for the first 10 hrs after nanocapsule administration, then after 24 hrs, and at euthanasia. Collected feces were pooled. Feces were weighted, resuspended in PBS at 37°C (0.1g of feces/1 mL of PBS) and centrifuged (2800 RCF, 5 mins, RT). Aliquots of 100 μL of urine and resuspended feces were transferred into 96-well black microplates. Fluorescence intensity was measured at 590 nm after excitation at 560 nm using Synergy H1 hybrid reader and analyzed with Gene5 Software (BIOTEK Instruments).
10
High-throughput Oncology Drug Screening
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A library of 133 FDA-approved oncology drugs was provided by the National Cancer Institute (NCI). Drugs were arrayed in polypropylene 384-well plates covering a 7-point concentration range (10 µM, 2.5 µM, 625 nM, 156 nM, 39 nM, 9.8 nM, 2.4 nM). Cells were suspended in RPMI 1640 Medium for SILAC (ThermoFisher Scientific) with 10% dialyzed FBS, 0.3 mM L-Lysine (Sigma), 1mM L-Citrulline (Sigma) with or without 1mM L-Arginine (Sigma) and plated at 1000 cells / well density. After 72 h, 50 µL of CellTiter-Glo reagent diluted 1:4 in PBS was added to each well and luminescence readings were performed using a Synergy H1 Hybrid Reader (Biotek). Each condition was assayed in duplicate (n=2) and % proliferation values were calculated by normalizing experimental wells to plate negative controls and averaging replicate values.
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