Collagen type 1

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

Collagen type I is a purified protein derived from bovine or rat sources. It is the most abundant form of collagen found in the human body, providing structural support for various tissues. This product is commonly used in cell culture and tissue engineering applications.

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Close spelling variants of this product

Collagen type I solution (4 citations) DQ™ type I collagen (16 citations) Rat type I collagen (2 citations) DQ®-FITC-conjugated type I collagen (2 citations) FITC-conjugated collagen type I (2 citations) Rat tail collagen type I (72 citations) Neutralized rat tail collagen type I (2 citations) Bovine collagen type I (17 citations) Collagen from rat tail (type I), (2 citations) Rabbit anti-type I collagen (2 citations)

104 protocols using collagen type 1

3D Collagen-Based Cell Invasion Assay

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Cell invasion rates were established using our previously published method
[18 (link),19 (link)]. Using a real-time cell imaging system (IncuCyte™ (Essen BioScience, Michigan, USA). In brief, 96-well plates were coated with a thin layer of collagen by transferring 300 μg/ml of collagen type I (Life Technologies™, Carlsbad, CA) and incubating at 37°C for 30 min. OVCAR-3 and SKOV-3 (2 × 10⁴ cells per well) were grown to confluence in complete growth media. Cell-free zones were created by generating a wound with a 96-Well WoundMaker™ (Essen BioScience, Michigan, USA). The cells were overlaid with 3 mg/ml collagen type I (Life Technologies™, Carlsbad, CA) and incubated at 37°C for 30 min to create a 3D matrix. Complete growth media was added on top of the layer of collagen. Cells were imaged automatically every 3 h over a time period of 48 h. The images were processed by the IncuCyte™ software package (Essen BioScience, Michigan, USA) to measure cell invasion by obtaining the Relative Wound Density (RWD, as defined by custom algorithms within the IncuCyte™ software package). These users informed algorithms identify the wound region and provide visual representations of the segmentation parameters. Image collection was created using three to five representative phase contrast images.

Kobayashi M., Salomon C., Tapia J., Illanes S.E., Mitchell M.D, & Rice G.E. (2014). Ovarian cancer cell invasiveness is associated with discordant exosomal sequestration of Let-7 miRNA and miR-200. Journal of Translational Medicine, 12, 4.

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Endothelial Cell Response to Neutrophil Extracellular Traps

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HCAECs (Lonza) were cultured on plates coated with 100μg/ml type 1 collagen (Thermo Fisher Scientific) in EGM-2 MV (Lonza) supplemented with 15% FBS (Gibco). After reaching confluence, HCAECs were conditioned either in 15% FBS or 15% baby rabbit serum (Cedarlane Corporation), decomplemented or not by treatment at 56°C for 30 minutes, and incubated with 1.5 mg/ml NETs for 6 hours. The supernatants were harvested for detached cells count and the attached cells were washed and fixed in 2% PFA for further immunocytofluorescence analysis.

Franck G., Mawson T.L., Folco E.J., Molinaro R., Ruvkun V., Engelbertsen D., Liu X., Tesmenitsky Y., Shvartz E., Sukhova G.K., Michel J.B., Nicoletti A., Lichtman A., Wagner D., Croce K.J, & Libby P. (2018). Roles of PAD4 and NETosis in Experimental Atherosclerosis and Arterial Injury: Implications for Superficial Erosion. Circulation research, 123(1), 33-42.

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Polyacrylamide Hydrogel Functionalization and Cell Adhesion

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Polyacrylamide (PAA) hydrogels were prepared as previously described (42 (link)). Briefly, PAA gel formation was initiated with ammonium persulfate (10% solution in ddH₂O; Sigma-Aldrich) and N,N,N′,N′-tetramethylethylenediamine (Sigma-Aldrich). Polymerized PAA gels were functionalized with sulfo-SANPAH (Invitrogen) and coated with type 1 collagen (200 μg/mL rat tail; Thermo Fisher). The Young’s modulus of the PAA gels was 8.55 ± 0.5 kPa. Cells were allowed to adhere to the gel for 4 to 6 h before flow cytometry.

Layton T.B., Williams L., Yang N., Zhang M., Lee C., Feldmann M., Trujillo G., Furniss D, & Nanchahal J. (2022). A vasculature niche orchestrates stromal cell phenotype through PDGF signaling: Importance in human fibrotic disease. Proceedings of the National Academy of Sciences of the United States of America, 119(13), e2120336119.

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Differentiation of Urine-derived Progenitor Cells into Podocytes

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Urine-derived renal progenitor cells (UdRPCs) of a 51-year-old male (UM51) were isolated as described in Rahman et al. [24 (link)]. Further, the UM51 cell line was immortalized with an hTERT-expressing plasmid (UM51 hTERT) [28 (link)]. The cell lines were cultured on 0.2% type 1 collagen (Thermo Fisher Scientific, Waltham, MA, USA) coated 6- or 12-well plates at 37 °C under hypoxic conditions. The cells were cultured in Proliferation Medium (PM) composed of 50% DMEM high-glucose (Gibco) and 50% keratinocyte growth basal medium (Lonza, Basel, Switzeland) supplemented with 5% fetal bovine serum (Gibco), 0.5% Non-Essential Amino Acid (Gibco), 0.25% Glutamax (Gibco), and 0.5% penicillin and streptomycin (Gibco). For further differentiation into podocytes, the cells were seeded at a low density (50,000 cells per 6-well) and cultured for 24 h in PM. On the next day, the medium was exchanged to Advanced RPMI 1640 (Gibco) supplemented with 0.5% fetal bovine serum, 1% penicillin and streptomycin, and 30 μM retinoic acid (Sigma-Aldrich Chemistry, Steinheim, Germany). After 7 days, the typical podocyte morphology was observed. Losartan (Sigma-Aldrich Chemistry) and ANG II (Sigma-Aldrich Chemistry) were diluted using Advanced RPMI 1640 to a final concentration of 100 μM ANG II or 1 μM losartan. First, the cells were incubated for 24 h with 1 μM losartan and then for 24 h with ANG II.

Thimm C., Erichsen L., Wruck W, & Adjaye J. (2023). Unveiling Angiotensin II and Losartan-Induced Gene Regulatory Networks Using Human Urine-Derived Podocytes. International Journal of Molecular Sciences, 24(13), 10551.

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Polyacrylamide Gels for Cell Culture

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Polyacrylamide gels with variable Young’s moduli were prepared according to a previously established protocol by Engler et al.40 . Briefly, acrylamide and bis-acrylamide mixture with indicated concentrations was allowed to polymerize on a glass slide, and the gel was then covered by sulfosuccinimidyl-6-[4′-azido-2′-nitrophenylamino] hexanoate (Sulfo-SANPAH; Pierce). After exposure to UV light for 10 min twice, the polyacrylamide sheet was washed twice and incubated with a solution of type I collagen (0.2 mg/ml; Gibco BRL, Gaithersburg, MD), type II collagen (0.01 mg/ml; Sigma-Aldrich, St. Louis, MO, USA), or matrigel (1:100; BD Biosciences, San Jose, CA, USA) overnight at 4 °C. The elasticity moduli of the soft and the stiff gels were 500 Pa and 10⁵ Pa, respectively.

Du J., Zu Y., Li J., Du S., Xu Y., Zhang L., Jiang L., Wang Z., Chien S, & Yang C. (2016). Extracellular matrix stiffness dictates Wnt expression through integrin pathway. Scientific Reports, 6, 20395.

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3D Cell Culture on Collagen and Gelatin

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The 3D culture experiments initially carried out using GFR Geltrex were repeated using type I collagen (1.5 mg/ml) (Gibco) and transglutaminase crosslinked gelatin (ColTgel) (101Bio) substrates. In the case of ColTgel, all three commercially available gel densities- soft (0.9-1.4 kPa), medium (14-20 kPa) and stiff (35-47 kPa) were tested. Both gels were cultured following the same 3D-on-top type culture protocol as that of GFR Geltrex gels.

Visakan G., Su J, & Moradian-Oldak J. (2021). Ameloblastin promotes polarization of ameloblast cell lines in a 3-D cell culture system. Matrix biology : journal of the International Society for Matrix Biology, 105, 72-86.

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Fibroblast Contractility Assay with Collagen Gels

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Fibroblast contractility was assessed by measuring changes in the surface area of type I collagen gels mediated by fibroblasts as previously described [37 (link)]. Serum-free DMEM was used in the assay to exclude the modulation of growth factors contained in serum [37 (link)]. The gels comprised type I collagen (Gibco, A10483-01; 0.75 mg/mL) and the cell suspension (5 × 10⁵ cells/mL) in HEPES-buffered DMEM (pH 7.4). Once the gels were set, cells were maintained in serum-free DMEM. TGF-β was added to the medium at a final concentration of 1 ng/mL. CNP was added to the medium at a final concentration of 1 μM every 24 h. After the fibroblasts were cultured in the gels for 3 days, the gels were released from the plate and the diameter of the gel was measured by using an image scanner connected to a computer running the public domain NIH image analyzing software (ImageJ version 1.48; available from http://imagej.nih.gov/ij). The percentage of contraction was calculated by using the formula 100 × (well diameter – gel diameter)/well diameter.

Kimura T., Nojiri T., Hino J., Hosoda H., Miura K., Shintani Y., Inoue M., Zenitani M., Takabatake H., Miyazato M., Okumura M, & Kangawa K. (2016). C-type natriuretic peptide ameliorates pulmonary fibrosis by acting on lung fibroblasts in mice. Respiratory Research, 17, 19.

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FT-IR Analysis of Collagen Structure

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The chemical structure characterization of PSP was conducted by infrared spectroscopy. The infrared spectra of PSP were measured using a 2000 Fourier-transform infrared spectroscopy (FT-IR) spectrophotometer (PerkinElmer, Waltham, MA, USA). Type I collagen (Gibco, Grand Island, NY, USA) was analyzed as a control. FT-IR analysis was performed at wavelengths of 4000 to 450 cm⁻¹.

Lee S.J., Lee J.H., Park J., Kim W.D, & Park S.A. (2020). Fabrication of 3D Printing Scaffold with Porcine Skin Decellularized Bio-Ink for Soft Tissue Engineering. Materials, 13(16), 3522.

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Isolation of Primary Mouse Vascular Smooth Muscle Cells

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The primary mouse VSMCs were isolated as described in the previous study (He et al., 2020 (link)). Briefly, the descending aorta was isolated from the 6-week-old male mice. Thereafter, the inner and outer layers of the vessel were removed by trypsin or microscissors. Arteries were then digested in 425 U/ml collagenase type II (Worthington, Cergy Pontoise Cedex France, Cat# 47D17411A) for 5 h at 37°C. Later, the cells obtained were resuspended in the basic culture medium. Then, VSMCs were seeded in a 25 cm² flask coated with 0.25 μg/cm² type I collagen (Gibco, Cat# A1048301). The VSMCs were verified by immunofluorescence, in which the smooth muscle marker was stained. The isolation of VSMCs was successful to reach 90% in cells (Supplementary Figure 1). Cells in the second passage were harvested for experiments.

Chen P., Hong W., Chen Z., Gordillo-Martinez F., Wang S., Fan H., Liu Y., Dai Y., Wang B., Jiang L., Yu H, & He P. (2022). CCAAT/Enhancer-Binding Protein Alpha Is a Novel Regulator of Vascular Smooth Muscle Cell Osteochondrogenic Transition and Vascular Calcification. Frontiers in Physiology, 13, 755371.

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Intestinal Tissue-on-a-Chip with Immune Cells

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Both chambers of the chip were coated with type I collagen (30 μg/ml; Gibco) and Matrigel (100 μg/ml; BD Biosciences, Bedford, MA, United States) in serum-free MEM, which was injected into the microchannels and incubated for 1 h before cell plating. Caco-2 cells (1 × 10⁵ cell/cm²) were stained with 5 mM green cell-tracker (CMFDA Dye, Invitrogen) were seeded in the lower microculture chamber, and the chip was turned upside down and incubated at 37°C allowing the seeded intestinal epithelial cells to grow on the membrane surface, Caco-2 static culture for 1 day, then continuously perfused into Caco-2 chambers at 60 μl/h using a multi-channel injection pump (LSP04-1A, LONGER Halma, England). After 2 days, HUVECs (1 × 10⁵ cells/cm²) stained with 5 mM red cell-tracker (CMPTX Dye, Invitrogen) were seeded into the upper microculture chamber and static culture for 1 day, then continuously perfused into all chambers at 60 μl/h using a multi-channel injection pump. After 3 days, PBMC (1 × 10⁷ cells/ml) with fresh antibiotic-free culture medium (50% HUVECs culture medium and 50% PBMC medium) was continuously perfused into the upper chamber (capillary side) at 60 μl/h using a multi-channel injection pump.

Zhao W., Yao Y., Zhang T., Lu H., Zhang X., Zhao L., Chen X., Zhu J., Sui G, & Zhao W. (2022). Primary exploration of host–microorganism interaction and enteritis treatment with an embedded membrane microfluidic chip of the human intestinal–vascular microsystem. Frontiers in Bioengineering and Biotechnology, 10, 1035647.

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