The protocol is identical to the one already described, with few modifications (Chan et al., 2007 (link)). U2OS cells grown in six-well plates were transfected for 48 h with control siRNA or siRNA targeting ATG13, ULK1, or FIP200. Subsequently, the medium was exchanged with the labeling medium (DMEM and 10% dialyzed FCS containing 0.2 µCi/ml [14C]-valine + 65 µM valine). After 18 h, the cells were placed into chase medium (DMEM, 10% FCS, and 2 mM valine) and incubated for additional 4 h to allow degradation of short-lived proteins. Cells were then transferred for 2 h in the EBSS medium to induce autophagy or in DMEM containing 10% FCS (no autophagy stimulation). The culture media were collected, and TCA was added to 10%. After centrifugation at 2,000 rpm for 10 min, the radioactivity in the soluble fraction (SF) was measured by scintillation counting. In parallel, cells were lysed in PBS containing 1% Triton-X and TCA was added to the lysate to 10% before freezing the samples overnight at −20°C. The radioactivity of TCA-soluble fraction (cytosol) as well as that of the TCA-insoluble pellet (pellet) dissolved in Solvable solution (PerkinElmer) was then measured after centrifugation at 2,000 rpm for 10 min. Protein degradation was determined as follows: (counts in SF + counts in cytosol)/(counts in SF + counts in cytosol + counts in pellet).
Solvable solution
Manufactured by PerkinElmer
Sourced in United States
Solvable solution is a laboratory product designed to facilitate the dissolution and preparation of samples for analysis. It is a versatile liquid formulation that aids in the solubilization of various compounds, enabling efficient sample preparation for various analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ultima gold scintillation mixture, by PerkinElmer (1 mentions)
Bicinchoninic acid protein assay kit, by Merck Group (1 mentions)
Fluorescence plate reader, by Molecular Devices (1 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (1 mentions)
Hionic fluor scintillation cocktail, by PerkinElmer (1 mentions)
Sodium phosphate dibasic, by Thermo Fisher Scientific (1 mentions)
Ethanol, by Avantor (1 mentions)
Sodium phosphate monobasic, by Thermo Fisher Scientific (1 mentions)
Carprofen rimadyl, by Zoetis (1 mentions)
Hydrogen peroxide, by Thermo Fisher Scientific (1 mentions)
Polyethylene glycol 300 peg300, by Merck Group (1 mentions)
2hp β cd, by Merck Group (1 mentions)
Heparin sodium salt, by Merck Group (1 mentions)
Ultima gold scintillation solution, by PerkinElmer (1 mentions)
Scintillation cocktail, by PerkinElmer (1 mentions)
Scintillation counter, by PerkinElmer (1 mentions)
9 protocols using solvable solution
1
Measuring Autophagy-Mediated Protein Degradation
2
Biodistribution of Radiolabeled Nanogels
3
Fluorometric Quantification of Microbial Uptake
4
Synthesis and Characterization of Radiolabeled E2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Carprofen (Rimadyl) was purchased from Zoetis (Parsippany, NJ, USA). Tritiated E2, dissolved in ethanol, was purchased from American Radiolabeled Chemicals (St. Louis, MO, USA). Solvable Solution and Hionic-Fluor scintillation cocktail were purchased from PerkinElmer (Waltham, MA, USA). Randomly methylated β-CD, 2HP-β-CD, β-CD, γ-CD, polyethylene glycol 300 (PEG300), heparin sodium salt, and non-radioactive E2 were purchased from Sigma-Aldrich (St. Louis, MO, USA), and ethanol was purchased from VWR (Radnor, PA, USA). Non-toxic black paint was purchased from Blick (Highland Park, IL, USA). Additional chemicals and solutions, including sodium chloride, 30% hydrogen peroxide, monobasic sodium phosphate, and dibasic sodium phosphate, were purchased from Fisher Scientific (Waltham, MA, USA).
5
Measuring Carrier-Mediated Glucose Uptake in Irradiated Mice
6
Tracing Brain Uptake of Na35SO4
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
400 μCi of Na235SO4 was diluted with PBS to 200 μl and injected into adult Slc13a4+/+ and Slc13a4+/− mice through the tail vein. 5 min following the injection, mice were euthanized to collect brain tissues (olfactory bulb and cerebellum were removed). 1 ml solvable solution (Perkin Elmer) was added to every 100 mg wet brain tissue in glass centrifuge tubes and incubated overnight at 37 °C. The next day, the tubes were gently shaken to homogenize the tissue and 100 μl 30% H2O2 was added to the tube. This step was repeated once when no visible bubble can be observed. 200 μl of the mixture was added to 1.5 ml scintillation cocktail (Perkin Elmer, USA) and placed at room temperature for 30 min before being analyzed in the scintillation counter (Perkin Elmer, USA) [34 (link)].
7
Quantitative Melanin Extraction from Tissue Equivalents
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Prior to photometric analysis, the samples of MelanoDerm™ (3 pieces of tissue equivalent per time point in experimental and control groups) and spheroids (256 spheroids per time point in experimental and control groups) were washed three times from the residues of the medium in PBS (pH = 7.4), after which they were stored at −20°C. To extract melanin, the samples were thawed and dried, and then 250 μl of Solvable solution (6NE9100, PerkinElmer) was added to each sample. Next, the samples were incubated for 18 h in a water bath at +60°C. After incubation, the samples were thoroughly mixed in a vortex mixer, undissolved particles were precipitated by centrifugation (5 min, 13,000 g), 100 μl of the supernatant was placed into a 96-well plate, and samples' optical densities were measured at a wavelength of 490 nm on a Multiscan GO plate photometer (Thermo Scientific, USA). A calibration curve was obtained by analyzing standard solutions with a known concentration of melanin prepared from dry matter (M863, Sigma-Aldrich). Measurements were performed in triplicate.
8
Quantifying Epidermal Melanin Content
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Regarding melanin content, epidermal tanned tissues were removed from the insert by cutting out the polycarbonate filter and then plunged into 400 μL of Solvable Solution (Perkin Elmer, Waltham, MA, USA). Then, samples were heated at 100 °C for 45 min, centrifuged, and melanin extract was measured by spectrophotometry at a 500 nm wavelength using a synthetic melanin calibration curve as reference.
9
Carmustine Wafer Dissolution and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Carmustine wafer extracted from the patient and unused carmustine wafer, Gliadel ® (mgi pharma ltd) were dissolved using 1 mL of solvable ® solution (Perkin Elmer). The solution was stirred overnight until complete dissolution. After filtration, solvable ® was evaporated and replace by CDCl3 before 1 H-NMR analysis. Carmustine, Bicnu ® (emcure pharma UK ltd) was directly dissolved in CDCl3 before 1 H-NMR analysis.
Analytical methods. 1
Analytical methods. 1
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!