Homogenized lungs were analyzed using a Soluble Collagen Assay Kit (BioVision, Milpitas, CA, USA), according to the manufacturer’s instructions.
Soluble collagen assay kit
Manufactured by Abcam
Sourced in United Kingdom, United States
The Soluble Collagen Assay Kit is a quantitative colorimetric assay used to measure the amount of soluble collagen in biological samples. The kit provides a simple, accurate, and reproducible method for determining the concentration of soluble collagen without the need for specialized equipment.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Pepsin, by Merck Group (1 mentions)
Clariostar plus, by BMG Labtech (1 mentions)
Fluorstar optima, by BMG Labtech (1 mentions)
Fibronectin, by Abcam (1 mentions)
Caspase 3 assay kit, by Thermo Fisher Scientific (1 mentions)
Phosphorylated p ampk, by Cell Signaling Technology (1 mentions)
α sma, by Abcam (1 mentions)
Total collagen assay kit, by Abcam (1 mentions)
Malondialdehyde mda assay kit, by Abcam (1 mentions)
Elisaplus kit, by Roche (1 mentions)
Compound c, by Selleck Chemicals (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Shrna, by Hanbio Biotechnology (1 mentions)
Cell counting kit 8 assay, by Merck Group (1 mentions)
Modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Insulin solution, by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
11 protocols using soluble collagen assay kit
1
Collagen Content Analysis of Homogenized Lungs
2
Quantifying Collagen in Mouse Lungs
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The amount of collagen in the mouse lung tissue was quantified using the Soluble Collagen Assay Kit (BioVision, Milpitas, CA, USA) according to the manufacturer’s protocol.
3
Collagen Quantification in Kidney Tissue
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5 mg of frozen kidneys were incubated overnight in 0.5 M acetic acid with 0.1 mg/ml pepsin (Sigma-Aldrich) at 4°C. Collagen quantification was performed with Soluble Collagen Assay Kit (Cat K532-100; BioVision) following manufacturer’s instructions. Fluorescence was recorded using the ClarioStar plus (Bmg Labtech), and data were normalized with total protein measured with Pierce BCA protein assay kit (Cat 23227; Thermo Fisher Scientific).
4
Quantifying Soluble Collagen from Cells
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The cells were collected in 1x PBS (pH 7.4) and centrifuged at 1000× g for 15 min and the cell pellet was suspended in cold 0.5 M acetic acid. The cell solution was homogenized on ice using a pre-chilled Dounce homogenizer. Following overnight incubation, the acidic solution was centrifuged at 10,000× g for 15 min at 4 °C. The supernatant was collected and neutralized using 0.5 M NaOH. Collagen concentration was measured using the Soluble Collagen Assay Kit® (ab241015, Abcam, Cambridge, UK) according to manufacturer’s instruction. The fluorescence was measured at an emission wavelength of 468 nm and an excitation wavelength of 376 nm using a Kaleido™ microplate reader. The graphs were plotted using GraphPad Prism 8.
5
Quantifying Serum Collagen Levels
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The serum collagen concentration was determined using the Soluble Collagen Assay Kit (ab241015, abcam). The fluorescence (Ex/Em = 340/460 nm) was measured in endpoint mode by a fluorometer (FluorStar Optima, BMG Labtech).
6
Comprehensive Metabolic Assays for Cellular Analysis
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Enzyme-linked immunosorbent assay (ELISA) assay including lactate dehydrogenase (LDH), creatine kinase MB isoenzyme (CK-MB), were purchased from MyBioSource (CA, United States). PKA (protein kinase A) colorimetric activity kit. Dihydrodichlorofluorescein diacetate (DCF-DA) and caspase-3 assay kit were obtained from ThermoFisher Scientific (CA, United States). Total antioxidant capacity (TAOC) assay kits, total superoxide dismutase (SOD) activity assay kits, malondialdehyde (MDA) assay kits, 3-nitrotyrosine (3-NT) assay kits, and 8-hydroxy 2 deoxy guanosine (8-OHdG) assay kits were purchased from Abcam (Cambridge, United Kingdom). ELISAPLUS kit was purchased from Roche Applied Science (Mannheim, Germany). Caspase 3/7 activity using a kit from Promega (WI, United States). Total collagen assay kit and soluble collagen assay kit were purchased from Abcam (Cambridge, United Kingdom). Phosphorylated (P−) AMPK, total (T−) AMPK, antibodies, and β-actin were purchased from Cell Signaling Technology (MA, United States). p53, cAMP, PKA, NRF2, HO-1, α-SMA, fibronectin, and phosphodiesterase 4D (PDE4D) were purchased from Abcam (Cambridge, United Kingdom). Compound C was obtained from Selleck Chemicals (Texas, United States). An AAV9 system harboring shPDE5D, shRNA, miR-223-3p mimic, miR-223-3p mimic inhibitor, or negative control was generated by HanBio Technology (Shanghai, China).
7
Exosome-Mediated Collagen and Wound Healing
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Collagen-I level was measured with a Soluble Collagen Assay Kit according to manufacturer instructions (ab241015, Abcam, Cambridge, UK). For wound healing experiments a scratch assay was used. Indicated cells were plated with ~80% intensity in 6-wells and after attachment, medium was changed after 24 h. Scratch was conducted in an appropriate size and cells were washed and treated with TC- or vSMC conditioned culture medium (CCM) from separate cell cultures, or with isolated exosomes resolved in appropriate cell culture medium. Images were conducted after 0, 12, and 24 h. Cell viability and cell proliferation was assessed using an EZ4U kit (Biomedica MP, Vienna, Austria) or cell counting kit (CCK)-8 assay (Sigma-Aldrich, Taufkirchen, Germany) according to manufacturer’s instructions. Cells were seeded in ~25% cell density and after attachment, cells were treated with indicated exosomes or vSMCs-control dissolved in vSMC culture medium. For EZU4 assay, the measurements were conducted 24 h. and 96 h. after treatment start. For CCK-8 assay, measurements were conducted 72 h. after treatment start. For cell growth analysis and collagen-I measurements, a recombinant human 20 ng PDGF-BB protein (ab79746, Abcam, Cambridge, UK) was used as positive control.
8
Cardiomyocyte Isolation and Characterization
9
Collagen Formation in Fibroblasts
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Soluble collagen assay kit (ab242291, Abcam, UK) was used to detect the collagen formation in 1 × 106 PdLFs treated with 1 µM, 10 µM, or 100 µM l -lactic acid in comparison to 1 µM PMA for 2 and 6 days. The experiment was conducted in triplicates, and the manufacturer’s instructions were followed.
10
Vaginal Epithelial Cells Collagen Assay
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Example 6
Methods: Between 1×104 and 1×108 human vaginal epithelial cells are seeded in complete growth medium (ATCC) overnight at 37° C., 5% CO2, and then transferred to Induction Buffer (Promega). Cells are either exposed to exosomes for 10, 30 or 60 minutes (between 3-200 rate per cell, e.g., 3, 7, 28, 142 or 200 rate per cell/exosomes per cell) or to forskolin (positive control) for 30 minutes (at 1, 10 or 100 μM).
Briefly, cells are resuspended in 2.5% acetic acid containing 0.1 mg/mL pepsin. Cells are disrupted on ice. The homogenate is sonicated on ice with a probe sonicator, and centrifuged at 12,000 g for 10 minutes. Supernatant is recovered and transferred to a new tube. Protein concentration is determined by a protein assay. The pH of the sample is neutralized by first adding 2N sodium hydroxide solution 1:6 directly on the sample. Then, 10×PBS is added 1:10 directly to the sample to the final concentration of 1×PBS. The sample is then used for collagen detection. Collagen detection assay is performed with Abcam's Soluble Collagen Assay Kit (ab242291) according to the manufacturer's instructions.
Results: Exosome-treated vaginal cells have increased collagen production. Exosomes are believed to modulate the matrix metalloproteinases and tissue inhibitor of matrix metalloproteinases-1 to cause an increase in collagen production.
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