RNA and DNA isolates were quantified utilizing the Quant-iT RiboGreen assay (Life Technologies-cat# R11490) and Quanti-iT PicoGreen assay (Life Technologies-cat# P7589), respectively. A total of 1 μL of RNA and RNA is required for quantification. Concentration is measured as ng/ul. For RNA quantification, isolates were excited at 485 ± 10 nm and the fluorescence emission intensity was measured at 530 ± 12 nm using a Victor X3 spectrophotometer (Perkin Elmer cat# 2030-0030). Fluorescence intensity was plotted versus RNA concentration over the calibration range, 0–100 ng/μL. For DNA quantification, isolates were excited at 480 nm and the fluorescence emission intensity was measured at 520 nm. Fluorescence intensity was plotted versus DNA concentration over the low calibration range, 0–50 ng/μL.
Victor x3 spectrophotometer
Manufactured by PerkinElmer
Sourced in France
The Victor X3 spectrophotometer is a high-performance multi-mode microplate reader designed for various applications in life science research. It is capable of absorbance, fluorescence, and luminescence detection across a wide range of wavelengths.
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Lab products found in correlation
Quant it ribogreen assay, by Thermo Fisher Scientific (1 mentions)
Quanti it picogreen assay, by Thermo Fisher Scientific (1 mentions)
Dmi6000b, by Leica (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Glucose assay kit, by Merck Group (1 mentions)
Colorimetric cell cytotoxicity assay kit, by Abcam (1 mentions)
Nuclear extraction kit, by Active Motif (1 mentions)
Imagequant las 4000 machine, by GE Healthcare (1 mentions)
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
8 protocols using victor x3 spectrophotometer
1
Quantitative Fluorescent Nucleic Acid Assay
2
Monitoring Lipolysis via Glycerol Quantification
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Lipolysis was monitored by quantifying glycerol in culture media at four different times (T = 0 h which corresponds to the start of the lipolytic treatments, after the 3 h of incubation to collect damaged cells contents, T = 15, 39, 63 h). Glycerol was measured using a glycerol assay kit (MAK117-AKT, Sigma-Aldrich) and Perkin Elmer Victor X3 spectrophotometer. Concentrations were calculated as μmoles per g of tissue.
3
Intracellular Nitric Oxide Quantification
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The Cell Meter™ Fluorimetric Intracellular Nitric oxide (NO) Activity Assay Kit- Trypsinised HUVECs were resuspended in medium at 5 × 105 cells/mL and incubated with 2 µL of 500× Nitrixyte™ Red (Component A) for 30 min at 37 °C. After washing in PBS, the cells were monitored for the fluorescence intensity at the FL4 channel (Ex/Em = 630/660 nm) using a FACSCalibur flow cytometer (BD, Mountain View, CA). The negative controls were cells without the Nitrixyte™ Red probe added. The data obtained were analysed by FlowJo software (Ashland, OR, USA).
The DAF-2DA fluorescence labelling technique- HUVEC cultures at confluence were washed with PBS and incubated with 5 µmol/L of DAF-2DA for 30 min. After incubation, cells were washed twice with PBS, and fixed with 2.5% PFA. Pictures were taken with an inverted fluorescent microscope (Leica DMI6000B, Leica Microsystems, Wetzlar, Germany) and intensity values were obtained with emission at 515 nm with a VICTOR X3 spectrophotometer from PerkinElmer Inc. (Waltham, MA, USA).
The DAF-2DA fluorescence labelling technique- HUVEC cultures at confluence were washed with PBS and incubated with 5 µmol/L of DAF-2DA for 30 min. After incubation, cells were washed twice with PBS, and fixed with 2.5% PFA. Pictures were taken with an inverted fluorescent microscope (Leica DMI6000B, Leica Microsystems, Wetzlar, Germany) and intensity values were obtained with emission at 515 nm with a VICTOR X3 spectrophotometer from PerkinElmer Inc. (Waltham, MA, USA).
4
Quantifying Iron Content in SPIO-Labeled Cells
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To determine the average content of iron per one SPIO-labeled cell, 5x105 labeled hMSCs were pelleted by centrifugation and dissolved by incubation in 0.5 ml of the iron release reagent (0.5 M HCl, 2.25% w/v KMnO4) for 2 hours at 60°C. 250 μl of the resulting solution were mixed with 125 μl of H2O and 25 μl of the iron detection reagent (6.5 mM of the sodium salt of 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4′′-disulfonic acid, 6.5 mM neocuproine, 2.5 М ammonium acetate, 1 M ascorbic acid). Light absorbance at 490 nm was measured with Victor X3 spectrophotometer (PerkinElmer) and iron concentration determined using the calibration curve created utilizing standard solutions with known iron content and expressed in pg iron per cell. Three independent experiments with separate cell preparations were carried out and results were presented as means of 3 experiments ± standard deviation.
5
Glucose Utilization Assay for FZ
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Glucose utilization was estimated in cells exposed to FZ using the commercially available glucose assay kit from Sigma as per manufacturer’s instructions. After 24 h of drug exposure, culture supernatants were collected and centrifuged to remove any cellular debris. Assay reagent was mixed with the culture supernatants and incubated at 25 °C for 30 min, after which the absorbance was recorded at 505 nm on a PerkinElmer VictorX3 spectrophotometer.
6
Colorimetric Cytotoxicity Assay Protocol
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Colorimetric Cell Cytotoxicity Assay Kit (Abcam#Ab112118, France) was used to estimate the cell viability. Briefly, cells were seeded in 96-well plate. After the realization of manufacturer’s instruction, absorbance was read at 570 and 605 nm using Victor™x3 spectrophotometer (Perkin-Elmer, France).
7
FOXO1 Activity Quantification Assay
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TransAM® FKHR/FOXO1 kit (Active Motif#46396, France) was used to estimate the FOXO1 activity. Briefly, at indicated time and condition, cells were harvested and used for a protein nuclear extraction using the Nuclear Extraction Kit (Active Motif#40410, France). For each point (technical duplicate and independent biologic triplicate), 15 µg of nuclear extract were used following the Active Motif’s instructions. ELIZA plate O.D. was read on a Victor™x3 spectrophotometer (Perkin-Elmer, France).
8
Protein Extraction and Western Blot Analysis
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For protein extraction, 1x10 6 cells were recollected by centrifugation and washed with PBS. Then, samples were lysated with RIPA buffer (150 mM NaCl, 1% NP40, 0.5% sodium deoxycolate, 0.1% SDS, 50 mM Tris pH 8), protease inhibitors (Complete Tablets Mini EDTA-free, Roche) and phosphatase inhibitors (10 mM NaF, 1 mM Na 3 VO 4 , 1µM DTT). Afterwards, samples were quanti ed using Bradford assay (Bio-Rad) and the absorbance at 595nm was quanti ed by using a Victor X3 spectrophotometer (Perkin Elmer).
Protein samples were resolved by SDS-PAGE gel electrophoresis, and they were transferred to a PVDF 0.45 µm membrane (Millipore) and blocked with 5% milk solution in Tris buffer solution (TBS). Membranes were incubated with the corresponding primary antibody (table 4) diluted in 3% BSA solution for 16 hours at 4ºC. After that, membranes were washed three times in TBS with 0,05% Tween-20 and were incubated with the corresponding secondary antibody. Finally, blots were revealed using Pierce ECL Plus Western Blotting Substrate (Thermo Fisher) and bands were obtained and quanti ed using ImageQuant LAS 4000 machine (GE Healthcare Life Science).
Protein samples were resolved by SDS-PAGE gel electrophoresis, and they were transferred to a PVDF 0.45 µm membrane (Millipore) and blocked with 5% milk solution in Tris buffer solution (TBS). Membranes were incubated with the corresponding primary antibody (table 4) diluted in 3% BSA solution for 16 hours at 4ºC. After that, membranes were washed three times in TBS with 0,05% Tween-20 and were incubated with the corresponding secondary antibody. Finally, blots were revealed using Pierce ECL Plus Western Blotting Substrate (Thermo Fisher) and bands were obtained and quanti ed using ImageQuant LAS 4000 machine (GE Healthcare Life Science).
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