Relative quantification of gene expression was determined by real-time RT-PCR. The expression of SMAD3 and SMAD7 genes was quantified using TaqMan Gene Expression assays as described previously [1 (link)]. Briefly, total RNA was isolated from freshly cut sections of cryopreserved jejunum previously embedded in OCT compound using the Purelink FFPE RNA isolation Kit protocol (Life Technologies). The total RNA was purified using RNA Clean and concentrator-25 Kit (Zymo Research, Irvine, CA, USA) with on-column DNase I treatment (Life Technologies) followed by RNA quantification using the Synergy H4 reader (Biotek, Winooski, VT, USA). Superscript III first-strand synthesis kit (Life Technologies) was used to synthesize cDNA from total RNA. TaqMan gene expression assays Rh02621726_m1, Hs00706299_s1, and Hs00178696_m1 (Life Technologies) were employed for the quantification of TGF-β, SMAD3, and SMAD7 transcripts, respectively, using an ABI 7900HT Fast PCR System (ThermoFisher Scientific). The expression of each gene was normalized against 18S rRNA expression using TaqMan 18S rRNA Endogenous Control Assay (Life Technologies). Relative gene expression was determined among different groups using the comparative threshold cycle (CT) method, and fold changes in the expression were evaluated using 2−ΔΔCT [33 (link)].
Synergy h4 reader
Manufactured by Agilent Technologies
Sourced in United States
The Synergy H4 reader is a multi-mode microplate reader from Agilent Technologies. It is designed to perform absorbance, fluorescence, and luminescence measurements in microplates. The Synergy H4 reader provides reliable and accurate data for a wide range of applications in life science research and drug discovery.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3 first strand synthesis kit, by Thermo Fisher Scientific (1 mentions)
Hs00178696 m1, by Thermo Fisher Scientific (1 mentions)
Taqman 18s rrna endogenous control assay, by Thermo Fisher Scientific (1 mentions)
On column dnase 1 treatment, by Thermo Fisher Scientific (1 mentions)
Rna clean and concentrator 25 kit, by Zymo Research (1 mentions)
Milli q advantage a10 ultrapure water purification system, by Merck Group (1 mentions)
24 well plate, by Sarstedt (1 mentions)
Prism 7, by GraphPad (1 mentions)
Hts tubulin polymerization assay kit, by Cytoskeleton (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Mouse il 6 quantikine elisa kit, by R&D Systems (1 mentions)
Mouse tnf α quantikine hs elisa kit, by R&D Systems (1 mentions)
Mouse fgf 21 quantikine elisa kit, by R&D Systems (1 mentions)
Celltiter blue reagent, by Promega (1 mentions)
Cladribine, by Selleck Chemicals (1 mentions)
Clofarabine, by Selleck Chemicals (1 mentions)
Bright glo luciferase kit, by Promega (1 mentions)
D luciferin, by Promega (1 mentions)
Ivis lumina 2, by PerkinElmer (1 mentions)
Recombiplastin, by Werfen (1 mentions)
18 protocols using synergy h4 reader
1
Quantifying SMAD3 and SMAD7 Gene Expression by RT-PCR
2
Quantifying Total Tau in Biological Fluids
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Instructions were followed according to those provided for the Thermo Fisher Human Tau (total) kit (Cat: KHB0041). Standard, Streptavidin-HRP, and wash buffer solutions were prepared according to the menu. For each well, 100 μl of standard and plasma or CSF sample (undiluted) were added, incubated overnight at 4°C with shaking, then washed 4 times with wash buffer. Detection antibody (100 μl/well) was then added, followed by incubation for 1 h at room temperature. Plates were washed 4 times with wash buffer, then 100 μl of diluted streptavidin-PE was added to each well, followed by incubation for 1 h at room temperature with shaking. Next, plates were washed 4 times, followed by addition of 100 μl of stabilized chromogen to each well. The reaction was allowed to occur for 10 min, then 100 μl of stop solution was added to each well, followed by plate reading on the BioTek Synergy H4 reader.
3
Phagocytosis Assay for PLGA Particles
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To assess the phagocytosis of PLGA particles, HA was labeled using a pHrodo Red Microscale Labeling Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. The labeling efficiency was calculated using a Synergy H4 reader (Biotek, Winooski, VT, USA) according to the manufacturer’s instructions. The labeled HA was then used for the preparation of PLGA particles, as described above for unmodified HA.
H1N1 influenza virus particles that were purified after sucrose gradient ultracentrifugation were labeled using the same method with a different molar ratio of pHrodo Red (6.125, 12.5, 50, 200 μM). The stain index (mean fluorescence/number of particles) of the virus and the PLGA particles was calculated. Virus particles with a stain index similar to the previously obtained PLGA NPs were chosen and normalized (1012/mL) using the NTA method prior to further experiments.
H1N1 influenza virus particles that were purified after sucrose gradient ultracentrifugation were labeled using the same method with a different molar ratio of pHrodo Red (6.125, 12.5, 50, 200 μM). The stain index (mean fluorescence/number of particles) of the virus and the PLGA particles was calculated. Virus particles with a stain index similar to the previously obtained PLGA NPs were chosen and normalized (1012/mL) using the NTA method prior to further experiments.
4
Metal Chelation Assay Protocol
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Metal chelation was determined in assay buffer (20 mM Na-HEPES, 150 mM NaCl, pH 7.4) according to the procedure described previously [42 (link)]. Stock solutions of the metals at 100 mM concentrations were prepared in MilliQ water (Milli-Q advantage A10 Ultrapure Water Purification System, Millipore, KGaA, Darmstadt, Germany). The FeCl2 stock solution (100 mM) was prepared in the presence of ascorbic acid (1 mM). Compound 4 at a concentration of 30 μM was incubated for 30 min with equimolar concentrations of CuCl2, ZnCl2, MgCl2, CaCl2, MnCl2, FeCl2, CoCl2, and NiCl2. Absorbance spectra were measured on a Synergy™ H4 reader (BioTek Instruments, Inc., Winooski, VT, USA).
5
Apoptosis Assay for Compound Screening
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At 24 h after seeding the cells in 24-well plates (Sarstedt, Nümbrecht, Germany), the tested compounds were applied at various concentrations. At the desired time points, the apoptosis rate was analyzed using a previously described procedure [27 (link)] with a Biotek Synergy H4 Reader. Camptothecin (1 μg/mL) applied for 4 h was used as a positive, technical control. Compounds at each concentration were tested at least three times. The obtained results were analyzed using GraphPad Prism 7.03.
6
Encapsulation Efficiency of Hyaluronic Acid in PLGA Nanoparticles
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Firstly, PLGA NPs (6 mg per sample) were lysed after resuspension in 1 mL of bio0.1 M NaOH and subsequent sonication in ultrasonic bath (Biosan, Rigas, Latvia) for 30 min. Neutral pH was restored by addition of 0.2M HCl prior measuring the encapsulation efficacy of HA in samples using the Bicinchoninic Acid Protein Assay Kit (AppliChem, Maryland Heights, MO, USA). According to the manufacturer’s instructions, 150 μL of samples were added to 96-well plates containing 75 μL Reagent A, 72 μL Reagent B, and 3 μL Reagent C. For an evaluation of the protein concentration, a calibration curve was prepared using 150 μL samples of HA in concentrations ranging from 1 to 250 μg/mL. A plate was incubated at 37 °C for 30 min. The absorbance of all the samples was measured at 562 nm using Biotek Synergy H4 reader.
7
Microtubule Polymerization Assay of Methyl Sulfone
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In vitro microtubule assembly was measured using an HTS-tubulin polymerization assay kit (Cytoskeleton, Inc., Denver, CO) with 96 half well plates. Tubulin was used at 40μM. Methyl sulfone was used at 0 (control), 0.8μM, 4μM, 40μM (equi-molar to tubulin), 400μM and 2mM. All samples were run in duplicate. Taxol (4μM) alone was also used as a control, and compared to Taxol (4μM) in the presence of 4μM methyl sulfone. Assembly buffer contained 1mM GTP, but no glycerol. Microtubule assembly was measured at BioTek, Inc., Winooski, VT, using a BioTek Synergy H4 reader with Gen5 data analysis software. The reader was set at 37°C, 340nm and a kinetic loop to read samples at one min intervals for 90 min. Initial lag time, Vmax, area under curve (AUC) and change in OD340 over 90 min were determined.
8
Quantifying Inflammatory Cytokines in Intestinal Cells
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Tissue samples were homogenized in lysis buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 10 mM EDTA and 1% Triton X-100 with a complete protease inhibitor cocktail (Roche)]. TNFα and IL6 levels in intestinal cell extracts were measured by the Mouse TNF-α Quantikine HS ELISA Kit (MHSTA50, R&D Systems) and Mouse IL-6 Quantikine ELISA Kit (M6000B, R&D Systems), according to the manufacturer's protocols. The results were analyzed using a Synergy H4 Reader (BioTek Instruments).
9
Quantitative p-tau ELISA Assay
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Instructions were followed according to those provided for the Thermo Fisher Human p-tau (pT231) phosphoELISA kit (Cat: KHB8051). Standard, anti-rabbit IgG HRP, and wash buffer solutions were prepared according to the menu. For each well, 100 μl of standard and plasma or CSF sample (undiluted) was added, incubated overnight at 4°C with shaking, then washed 4 times with wash buffer. Detection antibody (100 μl /well) was then added, followed by incubation for 1 h at room temperature. Plates were washed 4 times, then 100 μl of diluted anti-rabbit IgG HRP was added to each well, followed by incubation for 1 h at room temperature with shaking. Next, plates were washed 4 times, followed by addition of 100 μl of stabilized chromogen to each well to allow reaction to occur for 10 min. Then 100 μl of stop solution was added to each well, followed by plate reading on the BioTek Synergy H4 reader.
10
FGF-21 Levels in Aging Mice
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Mouse serum was harvested from both 3- and 5-month-old mice. There were at least 3 mice in each group. FGF-21 levels were measured by the Mouse FGF-21 Quantikine ELISA Kit (MF2100, R&D Systems) following the manufacturer’s instructions. The results were analyzed using a Synergy H4 Reader (BioTek Instruments).
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