PC concentrations were measured from tissue homogenates (i.e. liver, kidney, and brain). Oxidation or oxidative cleavage of proteins results in the production of carbonyl groups following standard technique [76 (link)], which covalently react with 2,4-dinitrophenylhydrazine (DNPH) to form 2,4-dinitrophenyl (DNP) hydrazone. DNP is detected via spectrophotometry at a wavelength of 370 nm [77 (link)]. Our study protocol differed by using 1ml of 20% TCA instead of 125μL of 50% TCA. Absorbances were read using a Spectramax M2 plate reader (Molecular Devices Corp., Sunnyvale, CA, USA). Samples were run in duplicate with a repeatability of r = 0.99 between control and samples. Protein content was determined using the Bradford assay using a bovine serum albumin (BSA) standard curve. 180 μL of guanidine-HCL solution was added to 20 μL of control sample (HCL solution) in a 1:10 ratio. Absorbances were read at 280nM using a Spectramax M2 plate reader (Molecular Devices Corp., Sunnyvale, CA, USA). Samples for the Bradford assay were run in duplicate with a repeatability of r = 0.99. The results are expressed in μMol per g protein.
Spectramax m2 plate reader
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax M2 plate reader is a multimode microplate reader designed for absorbance, fluorescence, and luminescence detection. It is capable of reading samples in 6- to 384-well microplates and can be used for a variety of applications in life science research and drug discovery.
Automatically generated - may contain errors
Lab products found in correlation
Cm h2dcfda, by Thermo Fisher Scientific (5 mentions)
Celltiter blue cell viability assay, by Promega (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
H2dcfda, by Thermo Fisher Scientific (4 mentions)
Bradford dye, by Bio-Rad (2 mentions)
Celltiter blue, by Promega (2 mentions)
Cm h2dcfda, by Molecular Devices (2 mentions)
Facsdiva, by BD (2 mentions)
Fm5 95, by Thermo Fisher Scientific (2 mentions)
20 ml plastic column, by Takara Bio (2 mentions)
Sepharose cl 2b, by Merck Group (2 mentions)
Microbca, by Thermo Fisher Scientific (2 mentions)
Qubit 4, by Thermo Fisher Scientific (2 mentions)
Evom2 voltohmmeter, by World Precision Instruments (2 mentions)
Transwell filter, by Corning (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (2 mentions)
Prism 9, by GraphPad (2 mentions)
Brdu cell proliferation kit, by Merck Group (1 mentions)
Firefly luciferase, by Merck Group (1 mentions)
130 protocols using spectramax m2 plate reader
1
Protein Carbonyl Quantification in Tissues
2
Histological and Cytokine Analysis of Gut Inflammation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The distal part of the gut was embedded in paraffin, sliced to a thickness of 6 μm, and stained with hemotoxylin and eosin (HE). The inflammation and tissue damage were observed in a blinded manner using a light microscope (80i, Nikon, Tokyo, Japan). The levels of cytokines (TNF-α, IL-6, IL-10, IL-12, and IFN-γ) in the gut tissues were measured using a respective enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen, Carlsbad, CA, USA) as inflammation indicators. The ELISA plates were read at a wavelength of 450 nm using a Spectramax plate reader (M2, Molecular Devices, San Jose, CA, USA).
3
Antioxidant Radical Scavenging Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The radical scavenging activities of eeRo and its fractions were determined as previously described by Brand-Williams et al., 1995 with modifications.16 This Kinetic method determine the antiradical activities of antioxidants based in reduction of free radical 2,2-Diphenyl-1-picrylhydrazyl (DPPH•). The reduction of radical DPPH• is observed with the decrease of absorbance at 518 nm at 0 min, 1 min and every 15 min until the reaction reached a plateau. Each fraction and the ascorbic acid were tested at concentrations of 1–300 μg/ml. DPPH•- was added to final concentration of 0.3 mM and allowed to react at room temperature for 30 min in dark conditions. The absorbance was measured using Spectra Max Plate Reader® M2 (Molecular Devices), Sunnyvale, California, USA.
4
Biomarkers of Cardiac and Oxidative Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serum Levels of troponin-I and creatinine kinase-MB (CKMB) were measured using special ELISA kits for rats (Cat. No. E4737 and Cat. NO. E4608, BioVision, CA, USA). Total levels of reduced glutathione (GSH) were measured using an assay kit (Cat. No. K454 (BioVision, CA, USA). Levels of interleukin-6 (IL-6) were measured using an ELISA kit (Cat. No. K4145, BioVision, CA, USA). Levels of superoxide dismutase (SOD) were measured using a rat’s assay kit (Cat. No. E458, BioVision, CA, USA). Levels of tumor necrosis factor-alpha (TNF-α) were determined by an ELISA kit (Cat. No., K1052, BioVision, CA, USA). Levels of malondialdehyde (MDA) were determined using an assay kit (Cat. No. K454, BioVision, CA, USA). ROS levels were measured using a special fluorometric kit (Cat. No. E-BC-K138-F, Elabscience, USA). The activity of PKR was measured using a special rat’s ELISA kit (Catalog No. LS-F16140, Lifespan BioSience Inc. (LSBio, Seattle, USA). The nuclear activity of NF-κB p65 was measured using an ELISA kit (Cat. No. Trans AM 40596, Active Motif, Tokyo, Japan). The absorbance and fluorescence signals in all analyses were measured by the M2 Spectramax plate reader (Molecular Devices, USA) or (FL600Bio fluorescent reader (Tek Instruments, Inc., USA). All analyses/kits were conducted for n = 6/group and following the manufacturer’s instructions.
5
Decellularization of Cadaveric Tissue ECM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cadaveric donor human tissues were obtained from a US-accredited tissue bank. ECM extraction and solubilization protocols were developed by Humabiologics®, Inc by employing a proprietary combination of physical, chemical, and enzymatic approaches. To evaluate the efficacy of the decellularization process, the remnant DNA from decellularized tissue ECMs was extracted using proteinase K solution at 37 °C for 24 h and quantified using Quant-iT Picogreen dsDNA kit (N = 3/group). Percent DNA removal was calculated by determining the total DNA amount in human tissues prior to decellularization using the same process. Briefly, 100 µL of solubilized tissue ECM before and after decellularization from each tissue source and equal volume of dsDNA reagent was added into individual wells of a 96-well plate. The plate was incubated at room temperature for 5 min by covering it with aluminum foil to protect it from light. Following this, fluorescence was measured at an excitation wavelength of 480 nm and emission wavelength of 520 nm using M2 Spectramax plate reader (Molecular Devices, San Jose, CA, USA). DNA concentration in each sample was calculated by generating a standard curve with known concentrations of DNA.
6
Quantifying Tissue-Derived ECM Gelation Kinetics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gelation kinetics assay was performed to determine the polymerization rate of ECM solutions derived from different tissue sources. Briefly, 100 µL of chilled neutralized ECM solution was casted into individual wells of a 96-well plate at 4 °C (N = 6/group) and inserted into M2 Spectramax plate reader (Molecular Devices) pre-heated at 37 °C. Absorbance was measured at 405 nm every 15 s for 60 min, with the absorbance value corresponding to the degree of fibril density in the hydrogel. All values were normalized between 0 and 1 for each sample of tissue ECM by utilizing a ‘min-max’ normalization technique using Equation (2). Polymerization rate was calculated by taking the slope of the linear region of the curve.
where X—set of absorbance values of x, X′—normalized value of X, min (X)—minimum value in X, max (X)—maximum value in X.
where X—set of absorbance values of x, X′—normalized value of X, min (X)—minimum value in X, max (X)—maximum value in X.
7
TGF-β Modulates Trypanosoma cruzi-Infected Cardiac Fibroblast Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CFs plated in a density of 1.5 × 104 cells per well in 96-well plates were infected with T. cruzi (Y strain, 10 parasites/host cell), and after 24 h of infection, the cultures were treated with 1, 5, and 10 ng/mL of TGF-β per 48 h. Measurement of proliferation of CFs was performed using BrdU Cell Proliferation Kit (Millipore) according to manufacturer’s instructions. Briefly, after 48 h of treatment with TGF- β and 72 h of T. cruzi infection, the cultures were incubated with 20 µg/mL of 5-bromo-2′-deoxiuridin (BrdU), allowing its incorporation in proliferating cells for 2 h. Cells were fixed, and the DNA was denatured with the solution provided by the kit for 30 min at room temperature. After washing, the BrdU-labeled DNA was detected by the monoclonal anti-BrdU antibody for 1 h at room temperature. The plate was washed, and the antigen–antibody complex was revealed by addition of peroxidase-conjugate goat anti-mouse IgG, antibody, and 3,3′,5,5′-Tetramethylbenzidine (TMB) as peroxidase substrate. The reaction was stopped with acid Stop Solution, and the colorimetric reaction was read in an M2 Spectramax Plate Reader (Molecular Devices, San Jose, CA, USA) at λ450 nm.
8
Denatured Luciferase Renaturation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine whether 17-AAG was biologically active and retained anti-Hsp90 activity at 39.5 °C, we used a standard denatured luciferase assay (48 (link)). Briefly, 250 nm firefly luciferase (Sigma) in denaturation buffer (25 mm Tris, pH 7.5, 8 mm MgSO4, 0.01% bovine γ-globulin, 10% glycerol) was incubated at 50 °C for 8 min, and 5-μl aliquots were cooled to room temperature for 3 min and then diluted to 25 μl in renaturation buffer (25 mm Tris, pH 7.5, 8 mm MgSO4, 0.01% bovine γ-globulin, 10% glycerol, 0.5 mm ATP, 5 mm KCl, 2 mm DTT). The 8E5/LAV cells at 37 and 39.5 °C, with or without 17-AAG, were gently lysed in the luciferase assay lysis buffer, and 25 μl was added to the heat-denatured luciferase reaction. The renaturation reaction was carried out at room temperature for 1 h, and 5 μl of the reaction was then added to 45 μl of the Steady-Glo luciferase substrate (Promega) in a white-coated 96-well plate with luminescence measured on an M2 SpectraMax plate reader (Molecular Devices) with normal speed and medium photomultiplier tube settings. Controls were performed identically in the absence of ATP and the renaturation step.
9
Quantifying EPAC1 GEF Activity
10
Catecholamine Measurement in Zebrafish
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Stress hormones epinephrine, norepinephrine, and their precursor dopamine were measured in fish (n = 12) using the 3-CAT ELISA (Rocky Mountain Diagnostics, Inc., Colorado Springs, CO) per manufacturer’s recommendations. Snap frozen whole zebrafish were ground in liquid nitrogen with mortar and pestle. To normalize variations in fish weight, the resulting whole fish powder was mixed with a buffer at a ratio of 100 mg fish powder to 500 μL HCL buffer solution, containing EDTA and sodium metabisulfite. Samples were centrifuged for 20 minutes at 10,000 × g at 4°C and supernatants collected. A standard curve was generated for each compound concentrations of 0.5, 1.5, 5, 20, and 80 ng/mL, for epinephrine and dopamine, and 0.2, 0.6, 2.0, 8.0, and 32.0 ng/mL for norepinephrine. Samples were diluted 1:1 to be in the range of the standard curve. A Spectramax® M2 plate reader (Molecular Devices, Sunnyvale, CA) was used to measure concentration at 450 nm.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!