A bovine serum albumin (BSA) solution at 1 mg/mL in PBS was used to prepare a protein standard curve ranging from 0 to 650 µg/mL. A total 10 µL of OMVs’ suspension was mixed with 2 µL of 1.5% Triton X100 (Sigma, St. Louis, MI, United States) and 13 µL of PBS. The mix was vortexed and incubated at room temperature for 5 min in order to solubilize the OMVs’ membrane. A mix constituted of 50 parts of bicinchoninic acid (BCA) (Sigma) and 1 part of copper II sulfate (Sigma) was prepared extemporaneously, and 200 µL of this mix were added to each well of a transparent, flat-bottom 96-well plate (Corning, Glendale, CA, United States). A total of 25 µL of a BSA standard solution or the OMV preparations were added in wells containing the BCA/Copper II mix. After incubation at 37 °C for 30 min to allow the development of the purple color, the absorbance of each well was measured at 536 nm using a Varioskan spectrophotometer (Thermofisher, Waltham, MA, United States). All the standard and sample solutions were prepared in triplicates.
Varioskan spectrophotometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Varioskan spectrophotometer is a versatile instrument designed for a range of spectroscopic measurements. It can perform absorbance, fluorescence, and luminescence analyses across a wide wavelength range. The Varioskan is capable of measuring a variety of sample types in multiwell plates, cuvettes, or other sample formats.
Automatically generated - may contain errors
Lab products found in correlation
Valinomycin, by Merck Group (2 mentions)
Disc3, by Merck Group (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Transparent flat bottom 96 well plate, by Corning (1 mentions)
Copper 2 sulfate, by Merck Group (1 mentions)
Sybr green pcr master mix, by Meridian Bioscience (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
Integra 400 plus, by Roche (1 mentions)
Tissue tearor, by Biospec (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Dcf diacetate, by Merck Group (1 mentions)
Pierce ldh cytotoxicity assay kit, by Thermo Fisher Scientific (1 mentions)
Mtt assay, by Merck Group (1 mentions)
Rpmi 1640 media, by Merck Group (1 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Disc3 5, by Merck Group (1 mentions)
Calpain glo kit, by Promega (1 mentions)
Oxiselect in vitro ros rns assay kit, by Cell Biolabs (1 mentions)
Quanti blue buffer, by InvivoGen (1 mentions)
21 protocols using varioskan spectrophotometer
1
Quantifying Extracellular Vesicle Protein Content
2
Colorimetric Assessment of ACC Deaminase Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For isolates that tested positive for ACC deaminase activity, their ACC consumption was assessed using a colorimetric ninhydrin assay as described by Li et al. (2015) (link). A total of 500 mg of ninhydrin and 15 mg of ascorbic acid were dissolved in 60 mL of ethylene glycol and mixed with 60 mL of 1M citrate buffer (pH 6.0) prior to use. Citrate buffer was prepared with 12.04 g sodium citrate dihydrate and 11.34 g citric acid in 1 L of distilled water and adjusted to pH 6.0.
A standard colorimetric calibration curve was performed with varying ACC concentrations. DF medium was prepared with respective ACC working concentrations of 3 mM, 2.5 mM, 2 mM, 1.5 mM, 1 mM, 0.5 mM, and 0 mM. Each ACC working solution (1 mL) was mixed with 2 mL of ninhydrin reagent in glass test tubes, which were capped, shaken and placed in a boiling water bath (100°C). After 15 min, the tubes were moved into a water bath at room temperature (∼20°C) for 2 min. The samples were then shaken for 30 s and left to stand at room temperature for 10 min to allow purple coloration to develop. The solution was transferred into a cuvette and absorbance measured at 570 nm with a Varioskan® spectrophotometer (Thermo ScientificTM).
A standard colorimetric calibration curve was performed with varying ACC concentrations. DF medium was prepared with respective ACC working concentrations of 3 mM, 2.5 mM, 2 mM, 1.5 mM, 1 mM, 0.5 mM, and 0 mM. Each ACC working solution (1 mL) was mixed with 2 mL of ninhydrin reagent in glass test tubes, which were capped, shaken and placed in a boiling water bath (100°C). After 15 min, the tubes were moved into a water bath at room temperature (∼20°C) for 2 min. The samples were then shaken for 30 s and left to stand at room temperature for 10 min to allow purple coloration to develop. The solution was transferred into a cuvette and absorbance measured at 570 nm with a Varioskan® spectrophotometer (Thermo ScientificTM).
3
HDAC Activity Assay in Jurkat Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Jurkat cells (1 × 106 cells) were synchronized for 18 h by serum deprivation, then treated with PaDef 47.3 μM and vehicle (DMSO 0.98%) for 6 h. Then, they were washed 2 times with cold PBS, and preparation of samples for the fluorometric assay was performed according to the manufacturer’s instructions [Histone Deacetylase (HDAC) Activity Assay Kit Fluorometric Kit ab156064, Abcam]. The changes in fluorescence intensity were monitored for 1 h in a Varioskan spectrophotometer (Thermo Scientific).
4
Quantifying T. cruzi DNA in Heart Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For evaluation of tissue parasitism, T. cruzi DNA was quantified in the heart samples by quantitative PCR (qPCR) analysis. DNA was isolated using a Wizard Genomic DNA purification kit (Promega, USA) according to the manufacturer’s instructions. DNA was quantified as a function of absorbance at 280 nm using a Varioskan spectrophotometer (Thermo Scientific, USA). qPCR analyses and DNA amplification were performed with SYBR green PCR Master Mix (Bioline, USA) using satellite DNA for T. cruzi Dm28c on an Applied Biosystems 7300 reverse transcription-PCR (RT-PCR) system (Thermo Fisher) using the following primers: forward, 5′-GCTCTTGCCCACAMGGGTGC-3′; reverse, 3′-CAAGCAGCGGATAGTTCAGG-5′. The parasitic load of T. cruzi in the cardiac tissues was calculated from a standard curve constructed using homogenized heart tissues enriched with 106 trypomastigotes of T. cruzi and serially diluted to provide a 7-log curve in a range of 10−1 to 105 parasite equivalents (par eq)/ml according to Duffy et al. (34 (link)). The limit of detection of the assay was 0.7 par eq/ml ( 95% confidence interval [CI], 0.0089 to 0.7925 par eq/ml) with a limit of quantification of 1.4 par eq/ml of homogenate (Fig. S2).
5
Biomarker Levels in Serum Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serum levels of IGF-1 were determined by ELISA in a Varioskan spectrophotometer (Thermo Scientific, Finland), following specific commercial assay protocol instructions (Chiron Corporation, USA).
The serum concentrations of glucose, triglycerides and cholesterol were determined by routine laboratory methods by a COBAS INTEGRA 400 Plus auto analyzer (Roche-Hitachi, Germany), Calibration Reagents (Roche) and Cassettes of the same brand.
The serum concentrations of glucose, triglycerides and cholesterol were determined by routine laboratory methods by a COBAS INTEGRA 400 Plus auto analyzer (Roche-Hitachi, Germany), Calibration Reagents (Roche) and Cassettes of the same brand.
6
Hepatic Oxidative Stress Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from whole liver tissues using RIPA buffer and quantified using the Bradford assay (Nanjing Jiangcheng Bioengineering Institute). The SOD, MDA and GSH-Px content of liver tissues was determined using the kits obtained from Nanjing Jiangcheng Bioengineering Institute, according to the manufacturer's protocols. To estimate hepatic ROS level, liver tissues were harvested and immediately homogenized in PBS using a Teflon homogenizer (Tissue-Tearor; BioSpec Products Inc.). Briefly, 50 µl liver homogenate was mixed with 4.85 ml of 100 mmol/l potassium phosphate buffer (cat. no. 700621-5; Cayman Chemical) and incubated with 2′,7′-dichlorofluorescin (DCF) diacetate (Sigma-Aldrich; Merck KGaA) in methanol at a final concentration of 5 µmol/l for 15 min at 37°C. The hepatic ROS level was determined as the amount of 2′,7′-dichlorofluorescein (DCF) quantified from a DCF standard curve. The BCA protein assay kit (Thermo Fisher Scientific, Inc.) was used to measure protein concentration. The fluorescence of DCF was measured with a Varioskan spectrophotometer (Thermo Fisher Scientific, Inc.) at excitation/emission wavelengths 480/530 nm.
7
3D Printed Constructs Cytotoxicity Assay
8
Evaluating rHDL-AuNP Effects on Glioblastoma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Glioblastoma cells (~5×103) were seeded in flat-bottom well plates, incubated for 24 hours at 37°C, and treated with 10% LPDS overnight and with rHDL or rHDL-AuNP. Following treatment, the morphology of the cells was examined by bright-field microscopy (Leica DM IRB inverted phase contrast microscope; Leica Microsystems, Wetzlar, Germany), and the images were recorded using a Retiga-2000R digital camera (Surrey, BC, Canada). Cell viability was assessed by MTT assay (Sigma-Aldrich). The cells were incubated with rHDL-AuNP at concentrations varying from 0.1 to 100 μg/mL in 10% LPDS for 24 hours at 37°C, followed by the addition of MTT dye for 4 hours at 37°C. The media were aspirated, and a solubilizing solution of 20% SDS/50% dimethylformamide at pH 4.7 was added to dissolve the formazan crystals. The absorbance was measured at 570 nm on a Varioskan™ spectrophotometer (Thermo Fisher Scientific). Percentage cell viability was calculated as follows: (absorbance of test runs/absorbance of control) ×100. All results reported are expressed as mean ± standard deviation (n=3) unless otherwise specified. Statistical analysis was carried out by using analysis of variance and Student’s t-test.
9
Measuring Cell Membrane Potential with DiSC3(5)
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cell transmembrane potential depolarization was measured using the membrane potential sensitive dye 3,3′-dipropylthiadicarbocyanine iodide, DiSC3 (5) (Sigma-Aldrich, St. Louis, MO, USA) [15 (link)]. For this, K562 cells were cultured in RPMI-1640 Media (Sigma) supplemented with 10% (v/v) fetal bovine serum (Corning) and 100 U/mL penicillin and streptomycin (Gibco, Waltham, MA, USA). Cells were grown in an atmosphere of 5% CO2 at 37 °C for 24 h. Then, K562 cells (1 × 105 cells/well) seeded in 96-well black-wall plates were incubated with 0.2 mM DiSC3 (5) (dissolved in Hanks’ HEPES buffer) for 30 min in a CO2 incubator. The fluorescence intensity was monitored for 2 h in a Varioskan spectrophotometer (Thermo Scientific, Waltham, MA, USA). DMSO 5% (Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control.
10
Measuring Membrane Potential Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In a 96-well plate with black-wall, 1 × 104 cells were seeded per well and incubated at 24 h. Previously incubated, cells were washed twice with Hanks-Hepes buffer and (200 μM) DiSC3(5) (3,3′-dipropylthiadicarbocyanine iodide, Sigma) per well and incubated for 30 min at 37 °C in CO2 as described (Guzmán-Rodríguez et al., 2016 (link)). Subsequently, 5 baseline fluorescence points were read at 625–670 nm and after reading, the treatments were added and reading was monitored for 2 h using a Varioskan spectrophotometer (Thermo Scientific). Valinomycin (444.52 µg/mL, Sigma) was used as a positive control for the change in membrane potential.
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!