Evans blue (20 mg/kg) was injected intravenously on day 6 p.i., and 3 h later, the mice were anesthetized and perfused, and the brains were removed and immediately frozen at −80°C for later processing. EB was quantified by a modified version of the previously described protocol (43 (link)). Briefly, EB was extracted from brains with one perfused brain per 2-ml skirted screw cap tube (Greiner). Seven hundred microliters of N,N-dimethylformamide (DMF) (catalog no. D4551; Sigma) was added with three silica beads (2.3 mm) (catalog no. 11079125z; Biospec) per tube and homogenized for 1 min at room temperature (Minibeadbeater-16 model 607; BioSpec Products). This homogenized solution was centrifuged at 16,000 × g for 20 min at 4°C. The supernatant was transferred to a separate tube and spun again at the same speed and temperature for 10 min. Two hundred microliters of this supernatant was then quantified in duplicate using a Varioskan Flash fluorometer (620-nm excitation; 695-nm emission; Thermo Scientific). For quantification, a standard curve was generated by using a uninfected perfused brain prepared in the same way and adding EB at a twofold dilution starting at 1 mg/ml to 1 µg/ml.
Varioskan flash fluorometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Varioskan Flash fluorometer is a versatile instrument designed for fluorescence-based measurements. It provides a range of detection capabilities, including fluorescence intensity, time-resolved fluorescence, and luminescence detection. The Varioskan Flash is capable of performing a variety of assays and can be used with a wide array of microplates, cuvettes, and other sample formats.
Automatically generated - may contain errors
Lab products found in correlation
Ac devd amc, by Merck Group (3 mentions)
Minibeadbeater 16 model 607, by Biospec (1 mentions)
Anti gfp antibody, by Takara Bio (1 mentions)
Anti dsred antibody, by Takara Bio (1 mentions)
Calbiochem, by Merck Group (1 mentions)
96 well black flat bottom plate, by Corning (1 mentions)
Evans blue, by Merck Group (1 mentions)
N n dimethylformamide (dmf), by Merck Group (1 mentions)
10 protocols using varioskan flash fluorometer
1
Quantification of Evans Blue in Brain
2
Detection of Serum Anti-DsRed Antibodies
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Serum anti-DsRed antibodies were detected using the Amplex ELISA Development Kit for Mouse IgG with Amplex UltraRed reagent (Life Technologies). Corning 96-well ELISA plates were coated with 10 μg/ml recombinant DsRed (Clontech) and incubated overnight at 4°C. The next day plates were washed 3 times with PBS containing 0.1% Tween-20 and blocked overnight at 4°C with PBS containing 1% bovine serum albumin. All serum samples were then diluted serially (3-fold dilutions) from 1:10 to 1:1,771,470 and incubated for 1 h at room temperature (RT) on the DsRed coated plates. An anti-DsRed antibody (Clontech) was used as a positive control for this assay and an anti-GFP antibody (Clontech) was used as a negative control. Afterward, plates were washed 3× with PBS/Tween-20 and incubated for 30 min at RT with 50 ng/ml of goat anti-mouse IgG horseradish peroxidase, washed 3× with PBS/Tween-20, and incubated for 30 minutes with Amplex Red at RT in a dark chamber. Fluorescence was quantified using a Varioskan Flash fluorometer (530 nm excitation; 590 nm emission) (ThermoScientific).
3
HDAC Inhibitory Potential of Quinoline-SAHA Derivatives
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The HDAC-inhibitory potential of 3ClQuin-SAHA and 3BrQuin-SAHA was deter-mined by using fluorogenic HDAC assay kits for the detection of pan-HDAC activity (Calbiochem, Merck Chemicals, Darmstadt, Germany) or the subtype-specific HDAC 2 and HDAC 6 activity (BPS Biosciences, San Diego, CA, USA). HDAC activity was measured according to the instructions of the supplier. SAHA (1–10 µM) served as positive control. Human HDAC enzymes derived from HeLa cell nuclear extracts (pan HDAC assay) or human recombinant HDAC-2 and HDAC-6 enzymes and adjacent fluorogenic HDAC substrates were used to determine HDAC activity levels. A 50 µL assay buffer containing 1 µg/µL bovine serum albumin, the human HDAC enzymes, 3ClQuin-SAHA (1–10 µM), 3BrQuin-SAHA (1–10 µM) or gefitinib (1–10 µM), and the corresponding HDAC substrates were added into a black 96-well assay plate. The reaction in each well was incubated at 37 °C for 30 min, followed by adding 50 µL HDAC developer reagent, and incubated at room temperature for an additional 15 min. Fluorescence intensity of the assay plates was measured on a Varioskan Flash fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) using an excitation wavelength of 360 nm and an emission wavelength of 460 nm.
4
Caspase-3 Activity Measurement Protocol
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Changes in caspase-3 activity were measured by the cleavage of the fluorogenic substrate AC-DEVD-AMC (EMD Millipore, Billerica, MA, USA), as described previously [43 (link)]. After incubation with SF5-SAHA or SAHA (1–10 µM) for 24 h the cells were harvested and lysed with lysis buffer. Subsequently, the lysates were incubated for 1 h at 37 °C with a substrate solution containing 20 μg/mL AC-DEVD-AMC, 20 mM HEPES, 10% glycerol and 2 mM DTT at pH 7.5. Substrate cleavage was measured fluorometrically using a Varioskan Flash fluorometer (Thermo Fisher Scientific, Waltham, MA, USA; filter sets: ex 360/40 nm, em 460/10 nm). n = 3 independent measurements were performed in triplicate, and data are given as the mean percentage increase ± SEM above control, which was set at 100%.
5
Fluorogenic HDAC Enzyme Inhibition Assay
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The ability of broxbam to inhibit HDAC1, -2, -4 and was measured using a cell free fluorogenic HDAC Assay (cat. nos. 50061, 50062, 50064, 50076 BPS Bioscience, Inc.). The HDAC activity was measured according to the protocols of the manufacturer. Briefly, purified human recombinant HDAC enzymes contained with the assay kit and fluorogenic HDAC substrates were used to measure HDAC activity. In total, 50 µl assay buffer containing 1 µg/µl BSA, the human recombinant HDAC enzyme, the test compound and the corresponding HDAC substrate was added into a black 96-well assay plate. The reaction in each well was incubated at 37°C for 30 min, followed by the addition of 50 µl HDAC developer reagent and incubation at room temperature for 15 min. Fluorescence intensity was measured using a Varioskan Flash Fluorometer (Thermo Fisher Scientific, Inc.) using an excitation wavelength of 380 nm and an emission wavelength of 460 nm.
6
Quantifying Vascular Leakage via Evans Blue
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To quantify vascular leakage, Evans blue (Sigma-Aldrich) was injected i.v. at a concentration of 20 mg/kg body weight diluted in PBS. After 4 h, brain and liver tissue were removed after perfusing mice with saline. Evans blue was extracted from tissues using N,N-dimethyl formamide (Sigma-Aldrich) as described previously (Kim et al., 2009 (link)). All samples were plated in triplicate on black 96-well flat-bottom plates (Corning), and fluorescence emission was quantified using a Varioskan Flash fluorometer (620-nm excitation; 695-nm emission; Thermo Fisher Scientific).
7
Caspase-3 Activity Measurement of Novel Compounds
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Changes in caspase-3 activity were measured by the cleavage of the fluorogenic substrate AC-DEVD-AMC (EMD Millipore, Billerica, MA, USA), as described previously [52 (link)]. After 24 h of incubation with 1a , 1b , 1c , 1e and sorafenib (1 and 10 µM) the cells were harvested and lysed with lysis buffer. Subsequently, the lysates were incubated for 1 h at 37 °C with a substrate solution containing 20 μg/mL AC-DEVD-AMC, 20 mM HEPES, 10% glycerol and 2 mM DTT at pH 7.5. Substrate cleavage was measured fluorometrically using a Varioskan Flash Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA; filter sets: ex 360/40 nm, em 460/10 nm). n = 3 independent measurements were performed in triplicate, and data are given as the mean percentage increase ± SD above control, which was set 100%.
8
Evaluating Cytotoxicity of Nanoparticles
9
Fluorometric Caspase-3 Activity Assay
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Changes in caspase-3 activity were measured by the cleavage of the fluorogenic substrate AC-DEVD-AMC (EMD Millipore, Billerica, MA, USA), as described previously [37 (link)]. After incubation with 3BrQuin-SAHA, 3ClQuin-SAHA, SAHA, or gefitinib (1–10 µM) for 6–48 h, the cells were harvested and lysed with lysis buffer. Subsequently, the lysates were incubated for 1 h at 37 °C with a substrate solution containing 20 μg/mL AC-DEVD-AMC, 20 mM HEPES, 10% glycerol, and 2 mM DTT at pH 7.5. Substrate cleavage was measured fluorometrically using a Varioskan Flash fluorometer (Thermo Fisher Scientific, Waltham, MA, USA; filter sets: ex 360/40 nm, em 460/10 nm).Three independent measurements were performed in triplicate, and data are given as the mean percentage increase ± SEM above control, which was set to 100%.
10
Cytochrome P450 Activity Assay in Fish
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The 7-ethoxyresorufin-O-deethylase (EROD) activity assay was conducted according to previous studies with slight modifications (e.g. buffer volume, employed basic instruments) [31 (link)]. The liver tissues of individual fish were homogenized in 0.25 M sucrose and centrifuged at 9,000 × g for 20 min at 4°C. To sample the microsomal fraction, the supernatant was transferred and centrifuged at 105,000 × g for 60 min at 4°C. Approximately 35 μg of microsomal fraction was transferred into the EROD buffer (0.1 M NaPO4, pH 7.6) with the addition of 7-ethoxyresorufin (ER) solution (0.4 mM ER in DMSO) and NADPH solution (10 mM NADPH in distilled water). The background subtraction was prepared with the same mixed buffer with the absence of the microsomal fraction. The resourufin production was measured with 530 nm excitation and 595 nm emission filters with a Varioskan Flash fluorometer (Thermo Fisher Scientific, Tewksbury, MA, USA). Results were represented as μmol min/mg protein.
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