To promote cell attachment, agarose substrates were functionalized with type I collagen using the bifunctional crosslinker, sulfo-SANPAH (Thermo Fisher Scientific, Waltham, MA)2 , 6 , 7 . Figure 1 presents a complete overview of the functionalized agarose substrate preparation setup. Excess water was removed prior to deposition of 100 μL of sulfo-SANPAH (1mg/mL) onto the surface of each film, followed by a 15 minute exposure to UV light. The darkened sulfo-SANPAH solution was aspirated and the procedure was repeated. The samples were thoroughly washed with DI water, and covered with a solution of 0.2 mg/mL bovine type I collagen (BD Bioscience, Franklin Lakes, New Jersey) and left undisturbed at 4 °C overnight. Following overnight incubation, the substrates were rinsed three times with 1× PBS and sterilized with UV irradiation before depositing cells.
Bovine collagen type 1
Manufactured by BD
Sourced in United States
Bovine collagen type I is a natural protein derived from bovine sources. It serves as a structural component in various biological applications.
Automatically generated - may contain errors
Lab products found in correlation
Sulfo sanpah, by Thermo Fisher Scientific (1 mentions)
Bacterial collagenase, by Merck Group (1 mentions)
0.4 μm filter, by Corning (1 mentions)
Egm mv, by Lonza (1 mentions)
Ga 1000, by Lonza (1 mentions)
Endothelial basal medium, by Lonza (1 mentions)
L wnt3a cells, by American Type Culture Collection (1 mentions)
L cells, by American Type Culture Collection (1 mentions)
Matrigel matrix growth factor reduced, by BD (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Alexa fluor antibody, by Thermo Fisher Scientific (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Rabbit monoclonal anti α sma antibody, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Human umbilical vein endothelial cells (huvec), by BD (1 mentions)
Human umbilical vein endothelial cells (huvec), by Lonza (1 mentions)
H1650, by American Type Culture Collection (1 mentions)
Egm mv bulletkit, by Lonza (1 mentions)
Trypsin solution from porcine pancreas, by Merck Group (1 mentions)
Human plasma fibronectin, by BD (1 mentions)
10 protocols using bovine collagen type 1
1
Functionalization of Agarose Substrates with Collagen
2
TIMP-2 Regulation of Apoptosis in Breast Cancer Cells
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MCF-7 cell transfectants were grown in medium containing 10% FCS; after 24 h (day 0) the medium was replaced with serum-free medium supplemented with TIMP-2 with or without the indicated additions. TIMP-2, U0126 or LY294002 were added every other day without changing the medium. The cells were collected at day 7 for Western blotting analysis of PARP degradation. 3D type I collagen gels (2 mg/ml) were made from a neutralized solution of bovine type I collagen (BD Biosciences, San Jose, CA, USA). Cells suspended in medium containing 10% FCS were added to the collagen mix prior to gelling, and gels were cast in 12-well plates. TIMP-2 was added every other day. The cells were recovered from 3D cultures at day 7 by dissolving the gels in 2 mg/ml bacterial collagenase (Sigma-Aldrich). The reaction was stopped with FCS, and the cells were immediately used for further analysis. MDA-MB-435 cells grown on glass coverslips were transiently transfected with MT1-MMP siRNA (as described below) 24 h and 48 h after seeding. Eight hours after the second transfection the culture medium was changed to serum-free medium with or without addition of TIMP-2 (100 ng/ml). TIMP-2 or an equivalent volume of control medium was subsequently added every 24 h in the following 2 days. The cells were then fixed and stained for characterization of apoptotic nuclei as described below.
3
Endothelial Cell Isolation from Tissue
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The tissue suspension was filtered with a 0.22 µm pore-size filter to obtain a monocellular cell suspension. Cells were plated into a 25 cm2 flask coated with bovine type I collagen (BD Biosciences, Milan, Italy) and cultured in endothelial proliferation medium, EndoPM, at 37 °C in 5% CO2 and 5% O2, according to published protocols [5 (link),21 (link),22 (link)], in order to isolate the endothelial cell population (ECs). The media was changed 1–2 times a week and passaged at a split ratio of 1:4 every 14 days [5 (link),9 (link)].
4
HUVEC culture with SWCNT aggregates
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HUVECs were used throughout this study and cultured as described previously.45 (link) HUVECs, endothelial basal medium (EBM), and supplements (EGM-MV: 5% fetal bovine serum, 12 μg/mL bovine brain extract, 1 μg/mL hydrocortisone, and 1 μg/mL GA-1000) were purchased from Lonza (USA). Cells were grown on 30 μg/mL bovine collagen type-I (BD Biosciences)-coated plates in a humidified incubator with 5% CO2 at 37 °C. A hypoxic chamber (Billups-Rothenberg Inc.) was filled with 2% O2 and placed within the tissue culture incubator. HUVECs were grown to passage 4 at ∼80% confluence and used for all experiments. Complete media consisted of EBM, 5% FBS, and all EGM-MV supplements, while starving media had reduced FBS levels of 0.5%. Serum-free media contained no additives. Conditioned media was made by incubating ∼80% confluent cells with starving media for 10 h. The resulting supernatants were filtered with a 0.4 μm filter (Corning, USA) to remove debris. Unless otherwise stated, aggregates were formed via introduction of (AT)15–SWCNT in 0.1 M NaCl at 2 μg/mL to test solutions and incubated under culture conditions for 6 h.
5
Immortalized Mouse Podocyte Cell Culture
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A conditionally immortalized mouse podocyte cell line was kindly provided by Professor Stuart Shankland and Mr Jeffery Pipping (Division of Nephrology, University of Washington, Seattle, WA, USA). Podocytes were maintained in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 0.1 mg/mL streptomycin, as previously described.28 (link) To initiate podocyte proliferation, cells were cultured at 33°C (permissive conditions) on bovine collagen type I (354231; BD Biosciences, Sydney, NSW, Australia) coated Primaria® cultureware 75 cm2 vented plastic flasks (353810; BD Biosciences). The medium was supplemented with 10 U/mL mouse recombinant interferon-γ (Roche Life Science, Sydney, NSW, Australia) to enhance expression of the temperature-sensitive large T antigen. To induce differentiation, podocytes were maintained at 37°C without interferon-γ (restrictive conditions) for at least 7 days, supplemented with 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin.
6
Cell Culture and Reagents for Breast Cancer Studies
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Mouse breast cancer cell lines EMT6 and 4T1, as well as L-cells and L-Wnt3a-cells were obtained from ATCC, USA. All cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen) containing 10% FBS (Hyclone). BD Growth Factor Reduced Matrigel, BD Matrigel Matrix Growth Factor Reduced, and BD BioCoat Control 8.0 micrometer PET Membrane 24-well Cell Culture Inserts were purchased from BD Biosciences, USA. ‘Super Special Grade’ C4S, C6S, CS-D, and CS-E, purified using Schiller’s column chromatographic method, were obtained from Seikagaku/The Associates of Cape Cod, USA. All CS preparations were received as lyophilized Na-salts, reconstituted in H2O, aliquoted, and stored at –20°C. Biological sources for the different CS types are as follows: C4S: sturgeon notochord; C6S: shark cartilage; CS-D: shark cartilage; CS-E: squid cartilage. Chondroitinase ABC (protease-free) was purchased from Seikagaku/The Associates of Cape Cod, USA. Bovine Collagen, Type I was purchased from BD Biosciences, USA. Wnt Antagonist I, IWR-1-endo was purchased from Calbiochem (EMD Millipore), San Diego, CA.
7
Transwell Migration Assay for ASC-HUVEC Interactions
8
Human and Murine Lung Cancer Cell Lines
9
Cell Proliferation Assay with FBS and Reagents
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Fetal bovine serum (FBS) was obtained from Innovative Research (Novi, MI). RPMI-1640 media with L-glutamine and without Sodium bicarbonate was purchased from HyClone Laboratories, Inc. (Logan, UT). CellTiter 96® AQueousOne Solution Cell Proliferation Assay (MTS) was purchased from Promega (Madison, WI). Antibiotic antimycotic solution (100X) stabilized, with 10,000 units penicillin, 10 mg streptomycin and 25 μg amphotericin B per ml (PSA) was obtained from Mediatech, Inc. (Manassas, VA). Human plasma fibronectin and bovine collagen type I was ordered from BD Biosciences (Franklin Lakes, NJ). Sodium bicarbonate, HEPES and trypsin solution from porcine pancreas were purchased from Sigma-Aldrich (St. Louis, MO). T-25 cm2 cell culture flasks were produced by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
10
Culturing 16HBE14o- Bronchial Epithelial Cells
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The 16HBE14o- cell line [75 (link)], a human bronchial epithelial cell line, was kindly provided by Dieter Gruenert (passage number P2.54; University California, San Francisco, CA, USA). Cells (passage number 11) were maintained in minimum essential media (MEM) 1x medium (with Earle’s Salts, 25 mM HEPES and without L-glutamine; Gibco BRL), supplemented with 1 % L-glutamine, 1 % penicillin/streptomycin and 10 % foetal calf serum. For experimental cultures, cells were seeded at a density of 0.5 x 106 cells/insert on transparent BD Falcon cell culture inserts (surface area of 0.9 cm2, pores with 3.0 μm diameter, PET membranes for 12-well plates). The cell culture inserts were pretreated with 150 μL fibronectin coating solution, containing 0.1 mg/mL bovine serum albumin (Sigma-Aldrich), 1 % bovine collagen Type I (BD Biosciences) and 1 % human fibronectin (BD Biosciences) in LHC Basal Medium (Sigma-Aldrich). Inserts were placed in BD Falcon™ tissue culture plates (12-well plates) with 1 mL medium in the upper and 2 mL in the lower chamber. The cells were kept at 37 °C in 5 % CO2 humidified atmosphere for 7 days (medium changed after 3–4 days).
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