Analogous segments of snap-frozen livers were lysed in RIPA buffer using a TissueLyser II (Qiagen, Germantown, MD, USA). Hepatic hydroxyproline content was evaluated using a colorimetric assay kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Liver lipids were extracted from liver samples using the Folch method [60 (link)]. Briefly, lysates were homogenized in methanol, and lipids were extracted by chloroform separation (methanol: chloroform (1:2)). After drying, the extracts were redissolved in 1% Triton-100, and cholesterol and triglyceride contents were measured using enzymatic colorimetric kits (Randox Lab. Ltd. Crumlin, UK) according to the manufacturer’s instructions. Biochemical parameters (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) in blood were evaluated on a Samsung PT10V clinical chemistry analyser. Plasma cholesterol and triglycerides were measured using enzymatic colorimetric kits (Randox Lab. Ltd., Crumlin, UK) according to the manufacturer’s instructions. Hepatic free iron content was determined using a colorimetric assay kit (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s instructions.
Colorimetric assay kit
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Hungary, Spain
The Colorimetric assay kit is a laboratory tool designed to quantify the concentration of a specific analyte in a sample through a color-based detection method. The kit provides the necessary reagents and protocols to perform the analysis, which relies on the sample's color change upon reaction with the kit's components. This kit can be used to measure the levels of various biomolecules, such as proteins, enzymes, or small molecules, in a variety of sample types.
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Lab products found in correlation
Multiskan go, by Thermo Fisher Scientific (4 mentions)
Celltiter glo luminescent cell viability reagent, by Promega (3 mentions)
Glutamine glutamate glo assay kit, by Promega (2 mentions)
Kim 1, by MyBioSource (1 mentions)
Statstrip xpress, by Nova Biomedical (1 mentions)
Ch 930 analyzer, by Siemens (1 mentions)
Albumin 201 system, by HemoCue (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Cnt prime 3d barrier culture medium, by CELLnTEC (1 mentions)
Antibiotic antimycotic 1x, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Tecan (1 mentions)
Quantikine rat il 6 and tnf α immunoassay kit, by R&D Systems (1 mentions)
Infinite 200 plate reader, by Tecan (1 mentions)
Sl 40r centrifuge, by Thermo Fisher Scientific (1 mentions)
Double beam uv spectrophotometer, by Shimadzu (1 mentions)
Colorimetric kit, by Merck Group (1 mentions)
Casp 3 c, by Merck Group (1 mentions)
Cayman nuclear extraction kit, by Cayman Chemical (1 mentions)
Alanine aminotransferase activity assay kit, by Merck Group (1 mentions)
Acyl coa oxidase based colorimetric kit, by Roche (1 mentions)
94 protocols using colorimetric assay kit
1
Comprehensive Liver Assessment Protocol
2
Comprehensive Kidney Function Assessment
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Serum creatinine was measured using an Atellica CH 930 Analyzer (Siemens Heathineers, Erlangen, Germany). To assess renal function, creatinine was also measured in urine samples according to the Jaffe principle. Urinary albumin concentration was measured using Hemocue®Albumin 201 System (HemoCue AB, Ängelholm, Sweden). Blood glucose (GlucoMen® Aero, A. Menarini diagnostics, Machelen, Belgium) and lactate levels (StatStrip Xpress, Nova Biomedical, Den Bosch, The Netherlands) were determined every 3 weeks until sacrifice. Plasma glucose was measured by using a colorimetric Assay Kit (Sigma-Aldrich, Saint Louis, MO, USA). At sacrifice, blood β-ketone levels were measured (GlucoMen® Aero 2K, A. Menarini diagnostics, Machelen, Belgium). Urinary NAG (Roche Diagnostics GmbH, Mannheim, Germany) and KIM-1 (MyBioSource, San Diego, CA, USA) were determined photometrically at the end of the study.
3
Colorimetric Assay of Serum ALT
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Serum ALT activity was determined to assess hepatocellular injury. ALT activity was measured using a colorimetric assay kit (Sigma Chemical) according to the manufacturer’s instructions. Briefly, the samples were centrifuged and stored in a freezer at -80°C until analysis. The enzyme activities of ALT in the sera were estimated using a microplate reader (BioTek, USA).
4
Epidermal Glucose and Lactate Metabolism
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Punch biopsies taken from the dorsal skin of the mice were trypsinized, and the obtained epidermal sheets were incubated in 250 μl CnT-Prime 3D Barrier culture medium (CELLnTEC Advanced Cell Systems) in duplicate at 37 °C and 5% carbon dioxide. After 24 hours, the medium was collected, deproteinized using a 10 kDa MWCO spin filter, and assayed for glucose and pyruvate concentration using a colorimetric assay kit (Sigma-Aldrich). The concentration of lactate in the medium was evaluated as previously described (Limonciel et al., 2011 (link)). Fresh medium was used in all the assays as a reference. Results were normalized by the total protein content of the tissue.
5
Hydroxyapatite Deposition in PLGA Scaffolds
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To determine non-cellular hydroxyapatite deposition leading to changes in calcium concentration in the medium, PLGA ± aCaP discs (3D meshes and 2D films, respectively) were incubated in 2 mL DMEM with 10% of FBS and Antibiotic-Antimycotic 1X (Thermo Fisher Scientific, Basel, Switzerland) for two weeks at 37 °C and 5% CO2, changing the medium every 3 or 4 days. A control was included using the medium without discs. Sample size was n = 3. The calcium concentration remaining in the solution of the samples was measured by a colorimetric assay kit (Sigma-Aldrich, Zurich, Switzerland) using a microplate reader (Tecan, Spark, Männedorf, Switzerland).
6
Intestinal Injury Biomarker Quantification
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Plasma intestinal fatty acid binding protein (I-FABP), IL-6, and TNF-α levels were measured using commercially available enzyme immunoassay kits from TSZ ELISA (TSZ Scientific, Framingham, MA, USA) and Quantikine Rat IL-6 and TNF-α immunoassay kit (R&D Systems, Minneapolis, MN, USA). Plasma D-lactate levels were measured using commercially available colorimetric assay kit from Sigma-Aldrich Inc. following manufacturer's instructions. Absorbances were measured at 450 nm.
7
Determination of NADPH/NADP+ Ratio
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The NADPH/NADP+ ratio was determined using a Colorimetric Assay Kit (Sigma-Aldrich). Briefly, 3 × 106 transfected cells were harvested and lysed with 200 μL NADP+/NADPH extraction buffer. Thereafter, the lysed cells were incubated for 5 min at 60 °C, and then 20 μL assay buffer and 200 μL counter NADPH/NADP+ extraction buffer were added. Next, the samples were centrifuged for 20 min, and the supernatants were used to determine the NADPH/NADP+ ratio. Finally, the absorbance at 565 nm was determined using a plate reader at 0 and 30 min.
8
Quantifying Lipid Peroxidation via MDA
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Malondialdehyde (MDA) was measured by using a colorimetric assay kit (Sigma-Aldrich Merck®, St. Louis, MO, USA) in plasma and urine samples as a marker of lipid peroxidation. The reaction of MDA with a chromogenic reagent was produced, generating a stable chromophore. Standards and samples were inserted in tubes with n-methyl-2-phenylindole in acetonitrile: Methanol (3:1) mixture. After that, HCl with 12N concentration was joined, and then the samples were incubated for 1 hour at 45 °C. Lastly, the absorbance was measured at 586 nm and, with a standard curve of known concentrations (0–20 nM), the MDA concentration was calculated.
9
Caspase-3 Activity Assay in Kidney and Cells
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Caspase-3 activity in the mice kidney and MDCK cells was measured using a colorimetric assay kit (Sigma-Aldrich) in accordance with the manufacturer’s protocol. In brief, kidney homogenates were incubated with the fluorometric caspase-3 substrate, Ac-DEVD-pNA, in the assay buffer. To account for nonspecific hydrolysis of the substrate, a control reaction mixture containing the caspase-3 inhibitor, acetyl-DEVD-CHO, in the assay buffer was used. Both mixtures were incubated for 90 min at 37 °C, with the absorbance being read at 405 nm.
10
Quantifying Mitochondrial Fumarase Activity
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For cco-1 single RNAi, glp-1 animals were bleach synchronized and grown at 25°C on bacteria expressing cco-1 dsRNA (or the empty vector control) until the first day of adulthood. For sequential double RNAi, glp-1 animals were bleach synchronized and grown at 25°C overnight on bacteria expressing tomm-20 dsRNA (or the empty vector control). Animals were then transferred to grow on bacteria expressing both cco-1 and tomm-20 dsRNA (or cco-1 dsRNA diluted to 1:1 ratio with bacteria containing the empty RNAi vector alone to match the strength in double RNAi treatment) until the first day of adulthood. Different fractions were separated via differential centrifugation. Proteins in each fraction were quantified with BCA assay, and an equal amount of protein from each fraction was used in fumarase assay. Fumarase activity was measured with a Colorimetric Assay Kit (MAK206; Sigma-Aldrich). The result was also confirmed with measuring fumarase activity in 100 mM potassium phosphate buffer with L-malic acid (malate) as substrate. Absorption at 240 nm was measured with a Tecan infinite 200 plate reader.
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