Bovine collagen 1

Manufactured by Merck Group

Sourced in United States

Bovine collagen I is a purified form of the primary structural protein found in bovine connective tissues. It is commonly used as a raw material in various cell culture and tissue engineering applications.

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6 protocols using bovine collagen 1

Collagen Quantification and Scaffold Visualization

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Collagen production (n=5) was quantified with a modified hydroxyproline assay[30 (link)]. Briefly, the samples were first desiccated (CentriVap, Labconco, Kansas City, MO) and digested for 18 hours at 65°C in a solution of 20 μL/mL papain (Sigma-Aldrich), buffered in 0.1 M sodium acetate, 10 mM cysteine HCl, and 50 M ethylenediaminetetraacetate. A 250 μl aliquot of the digest was concentrated by desiccation and samples were subsequently hydrolyzed in 10μl of 10N NaOH and autoclaved for 25 min. The hydrolysate was then oxidized by a buffered chloramine-T reagent for 25 min before the addition of Ehrlich's reagent. Sample absorbance was measured at 550 nm (Tecan), and collagen content was obtained by interpolation along a standard curve generated using bovine collagen I (Sigma-Aldrich).
Matrix and cell distribution (n=2) was also visualized using hematoxylin and eosin (H&E, Sigma-Aldrich) and Picrosirius red stains[31 (link)]. Briefly, samples were fixed overnight in a 10% neutral buffered formalin containing 1% cetylpyridinium chloride (Sigma-Aldrich) solution, and subsequently stored in 0.01 M cacodylic acid (Sigma-Aldrich) until sectioning. Scaffolds were embedded in 5% poly vinyl alcohol (Sigma-Aldrich) frozen sectioning medium, and cut in cross-section to 10 μm thickness using a cryostat (Bright Instrument Company, Cambridgeshire, England).

Lee N.M., Erisken C., Iskratsch T., Sheetz M., Levine W.N, & Lu H.H. (2016). POLYMER FIBER-BASED MODELS OF CONNECTIVE TISSUE REPAIR AND HEALING. Biomaterials, 112, 303-312.

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VEGF-Induced Cell Migration Assay

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A QCM 24-Well Colorimetric Cell Migration Assay Kit was used to perform migration assay. Both sides of inserts were coated with 0.1 μM of bovine collagen I (C4243, Sigma-Aldrich) for 1 h at 37 °C. Then, the inserts were washed with water, dried in a laminar flow cabinet, and disposed on 24-well cell culture plate covers. Solutions containing 30 ng/mL of VEGF-A165 preincubated with or without 0.1 μM of LAMA3_3043–3067 peptide in a medium (MCDB-131, 0.05% BSA) were added to the bottom side of the transwell (500 μL/well). Directly thereafter, HUVEC cells (Lonza) in a medium containing 0.05% BSA (300 μL/transwell, 4 × 10⁴ cells/transwell) were added to the transwell upper parts. After 6 h, migrated cells were stained and absorbance at 560 nm was measured according to the manufacturer’s instructions.

Ishihara J., Ishihara A., Fukunaga K., Sasaki K., White M.J., Briquez P.S, & Hubbell J.A. (2018). Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing. Nature Communications, 9, 2163.

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Chemokine-Guided BMDC Migration Assay

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Migration of BMDCs to a chemokine gradient was assessed as previously described by Haessler et al. (39 (link)). Briefly, BMDCs derived from transgenic mice expressing a fluorescent tag were stimulated with IC for 24 hours in the presence of iBET or DMSO control. iBET-treated cells were ECFP labelled and DMSO-treated cells EGFP labelled. After washing, cells were resuspended to a concentration of 10 × 10⁶ cells/mL and incorporated within 300 μL of a 1.5 mg/mL bovine collagen gel (20 μL 10x Minimum Essential Media Eagle (Sigma), 20 μL water, 10 μL 7.5% sodium bicarbonate solution (Sigma), 50 μL RPMI 1640, 150 μL 3mg/mL bovine collagen I (Sigma), 50 μL cell suspension in RPMI) loaded into a μ-Slide Chemotaxis chamber (Ibidi), according to the manufacturer’s instructions. The chamber was incubated for 15 minutes at 37°C, inverting once to ensure uniform cell distribution during collagen gelatination. Following incubation and solidification of the gel matrix, CCL19 (100 ng/mL, Peprotech) was applied to the chemoattractant solution reservoir and the sink reservoir was maintained base media. Migratory behaviour was imaged using time lapse confocal microscopy on an inverted Zeiss LSM 780 inverted confocal microscope with a 20X 0.9 NA objective, with stacked images taken every 45 to 60 seconds, over 2 to 4 hours.

Banham G.D., Lee C.Y., Ferdinand J.R., Matthews R.J., Jing C., Smithers N., Prinjha R.K, & Clatworthy M.R. (2022). Bromodomain Inhibitors Modulate FcγR-Mediated Mononuclear Phagocyte Activation and Chemotaxis. Frontiers in Immunology, 13, 885101.

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3D Collagen Gel Contraction Assay

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Serum starved ASM cells were suspended in a neutralized solution of bovine collagen-I (Sigma-Aldrich; C4243) and 10x DMEM (Sigma-Aldrich) and cast in 24-well tissue culture plates to create 7 mm-thick three dimensional (3D) collagen gels. Gels were allowed to set for 24 hours before being overlaid with DMEM. After 16 hours, gels released from wells and transferred to 6-well plates containing DMEM/F12 (Sigma-Aldrich) [36] (link). Contraction was stimulated with bradykinin (Sigma-Aldrich). Gel contraction was quantitated over 60 minutes using Genesnap camera (Genetools, Syngene, and Cambridgeshire, UK) with the reduction in initial gel area quantitated using Image J (http://rsb.info.nih.gov/ij). In experiments involving MMP inhibition, gels were treated with 10 µM ilomastat (Sigma-Aldrich) 1 hour prior to bradykinin stimulation.

Rogers N.K., Clements D., Dongre A., Harrison T.W., Shaw D, & Johnson S.R. (2014). Extra-Cellular Matrix Proteins Induce Matrix Metalloproteinase-1 (MMP-1) Activity and Increase Airway Smooth Muscle Contraction in Asthma. PLoS ONE, 9(2), e90565.

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Collagen Extraction and Purification

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Bovine collagen-I and human collagen-III were purchased from Sigma (MO, USA). Human collagen-I was obtained from Elastin Products (MA, USA). Human collagen-II was from Millipore (MA, USA). Collagenase from Clostridium hystolyticum was purchased from Nacalai Tesque (Kyoto, Japan). OPA was from Alpha Aesar (MA, USA). 3,4-DHPAA was purchased from TCI (Tokyo, Japan). Boric acid and NaIO₄ were obtained from Wako Pure Chemicals (Osaka, Japan). Peptides and non-collagenous proteins were purchased from Sigma, Wako Pure Chemicals, or Bachem (Bubendolf, Switzerland).

Yasmin H., Kabashima T., Rahman M.S., Shibata T, & Kai M. (2014). Amplified and selective assay of collagens by enzymatic and fluorescent reactions. Scientific Reports, 4, 4950.

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Apoptosis Induction Assay in BEAS-2B and HTBE Cells

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To measure the induction of cell death by drug treatment, BEAS-2B or HTBE cells were seeded in 96-well plates in appropriate culture media. For BEAS-2B cells, plates were prepared in advance by coating with a solution of bovine collagen I (0.03 mg/ml; Sigma-Aldrich, #804592) in 0.02 N of acetic acid for 1 hour at room temperature and then by rinsing with PBS. Cells were seeded at a density of 5000 cells per well. The day after seeding, seeding media was removed and replaced with media containing indicated doses of bortezomib, BH3 mimetics, or DMSO vehicle control. Drug treatment was carried out for 48 hours, and then cells were trypsinized for staining and FACS analysis. To stain for markers of apoptosis, Alexa Fluor 488–conjugated annexin V (0.5 μg/ml; gift from T. Letai) and PI (1 μg/m; Thermo Fisher Scientific, #BMS500PI) were added to 10X annexin V binding buffer [0.1 M Hepes (pH 7.4), 1.4 M NaCl, and 25 mM CaCl2 solution, sterile filtered]. Ten microliters of 10X annexin/PI solution was added to 100 μl of cell suspension, and the mixture was incubated on ice away from light for 20 min. After staining, cells were analyzed immediately by flow cytometry using an Attune NxT cytometer.

Inde Z., Croker B.A., Yapp C., Joshi G.N., Spetz J., Fraser C., Qin X., Xu L., Deskin B., Ghelfi E., Webb G., Carlin A.F., Zhu Y.P., Leibel S.L., Garretson A.F., Clark A.E., Duran J.M., Pretorius V., Crotty-Alexander L.E., Li C., Lee J.C., Sodhi C., Hackam D.J., Sun X., Hata A.N., Kobzik L., Miller J., Park J.A., Brownfield D., Jia H, & Sarosiek K.A. (2021). Age-dependent regulation of SARS-CoV-2 cell entry genes and cell death programs correlates with COVID-19 severity. Science Advances, 7(34), eabf8609.

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