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Purcol

Manufactured by Advanced BioMatrix
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PurCol is a Type I collagen product derived from bovine sources. It is a highly purified, sterile collagen solution suitable for a variety of cell culture and tissue engineering applications.

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Lab products found in correlation

Dq type 1 collagen, by Thermo Fisher Scientific (2 mentions) Mmp 8, by R&D Systems (2 mentions) Mmp 13, by Thermo Fisher Scientific (2 mentions) Collagenase 5, by Merck Group (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Human mature vegf c, by R&D Systems (2 mentions) Anti vegfr2, by Cell Signaling Technology (2 mentions) Duolink assay, by Olink (2 mentions) Insulin transferrin selenium sodium pyruvate, by Thermo Fisher Scientific (1 mentions) Fugene 6, by Promega (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Rs2000, by Rad Source (1 mentions) Trypsin edta, by Thermo Fisher Scientific (1 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions) Transwell chamber, by Corning (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

PurCol bovine collagen

14 protocols using purcol

1

Quantifying Collagenase Activity in Wound Samples

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Wound and explant samples were placed in the upper chambers of transwells in 50 µl, and 1% (v/v) quenched fluorescein-conjugated DQ™ type I collagen (1 µg/ml; Life Technologies, Grand Island, NY) was added to the bottom chambers in 500 µl of phenol red-free RPMI with 1% FBS and 1% penicillin/streptavidin. Collagenase activity was defined as the fluorescence detected in the bottom chamber (excitation 480 nm, emission 530 nm) minus fluorescence in blank controls (no sample in the upper chamber). For 3D matrix, we mixed native type I collagen (PurCol, Advanced BioMatrix, Carlsbad, CA) with DQ collagen and seeded explants onto the mixed matrix and assessed collagenase activity. For antibody depletion, we cultured samples with antibodies against MMP-8 (R&D Systems, Minneapolis, MN) or MMP-13 (Thermos Fisher Scientific, Waltham, MA). For macrophages, cells were incubated with phenol red-free RPMI containing 1% DQ collagen and 6 h.
Rohani M.G., McMahan R.S., Razumova M.V., Hertz A.L., Cieslewicz M., Pun S.H., Regnier M., Wang Y., Birkland T.P, & Parks W.C. (2015). MMP-10 Regulates Collagenolytic Activity of Alternatively Activated Resident Macrophages. The Journal of investigative dermatology, 135(10), 2377-2384.
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2

Genetically Engineered PDAC Cell Line

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KPsh cells (kindly donated by Scott W Lowe) derive from PDAC that developed in a Pdx1-cre;LSL-KrasG12D;Col1a1-TRE-shp53-shRenilla;Rosa26-CAGs-LSL-rtTA-IRES-mKate2 mice [25 (link)]. Cells were grown in DMEM (10% FBS Gibco; penicillin–streptomycin 2 units/ml) at 37 °C, 5% CO2 and they were propagated on collagen-coated plates (PurCol, Advanced Biomatrix, 0.1 mg/ml). KPsh were maintained in 1 μg/ml doxycycline to keep off p53 through doxycycline-dependent shRNA against Trp53; to allow p53 expression the doxycycline was removed 48 h before further procedure. KPsh cells have been authenticated as described in the source papers (references above); we also confirm p53 status by RT-qPCR or western blot.
Panatta E., Butera A., Celardo I., Leist M., Melino G, & Amelio I. (2022). p53 regulates expression of nuclear envelope components in cancer cells. Biology Direct, 17, 38.
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3

Quantitative Analysis of cGAMP Signaling

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293T cGAS ENPP1−/− cells were plated in plates coated with PurCol (Advanced BioMatrix). In some experiments, the cells were transfected with indicated plasmids complexed with Fugene 6 (Promega) 24 hours prior to the export experiment. At the start of the experiment, the media was replaced with serum-free DMEM supplemented with 1% insulin-transferrin-selenium-sodium pyruvate (ThermoFisher) and 100 U/mL P/S. At indicated times, the media and cells were removed and centrifuged at 1000 rcf for 10 minutes at 4 °C. Cells were lysed in 30 to 100 μL of 50:50 acetonitrile:water supplemented with 500 nM internal standard and centrifuged at 15,000 rcf for 20 minutes at 4 °C. Media was supplemented with internal standard at 500 nM and 20% formic acid. If media cGAMP enrichment was necessary, the media was acidified with 0.5% acetic acid, supplemented with internal standard, and applied to HyperSep Aminopropyl SPE columns (ThermoFisher Scientific) as described previously22 (link). Eluents were evaporated to dryness and reconstituted in 50:50 acetonitrile:water. The media and cell extract were submitted for mass spectrometry quantification of cGAMP, ATP, and GTP.
Carozza J.A., Böhnert V., Nguyen K.C., Skariah G., Shaw K.E., Brown J.A., Rafat M., von Eyben R., Graves E.E., Glenn J.S., Smith M, & Li L. (2020). Extracellular cGAMP is a cancer cell-produced immunotransmitter involved in radiation-induced anti-cancer immunity. Nature cancer, 1(2), 184-196.
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4

Preparation of Irradiated 3T3-J2 Feeder Cells

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Swiss 3T3 mouse fibroblast (J2 strain) feeder cells [23 (link), 24 (link)] were prepared as previously described [15 (link)]. Briefly, live 3T3-J2 cells were maintained in a 37 °C incubator with 5% CO2 in Complete Medium (DMEM high glucose containing 10% FBS (Gemini, Sacrament, CA), 100 μg/mL penicillin, 100 μg/mL streptomycin, and 100 μg/mL Glutamax (Life Technologies, Carlsbad, CA)). Feeder cells were removed from flasks at 80–90% confluence using 0.05% trypsin-EDTA (Life Technologies, Carlsbad, CA), re-suspended in Complete Medium at a concentration of 5 × 106 cells per ml and irradiated at 30 Gy in an RS-2000 (Rad Source, Suwanee, GA) machine. Irradiated 3T3-J2 cells were plated onto Purcol (Advanced Biomatrix, Carlsbad, CA) coated dishes to achieve 80% confluence and were allowed to attach for at least 4 h before the addition of intestinal epithelial cells.
Moorefield E.C., Blue R.E., Quinney N.L., Gentzsch M, & Ding S. (2018). Generation of renewable mouse intestinal epithelial cell monolayers and organoids for functional analyses. BMC Cell Biology, 19, 15.
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5

Collagen Scaffold Preparation Protocol

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The collagen scaffold was prepared by using the method described previously13. Briefly, a purified bovine collagen solution (PurCol; Advanced BioMatrix Inc., Tucson, AZ, USA) and 10×PBS were mixed in an 8:1 ratio and adjusted to a pH of 7.4 with 1 mL of sodium hydroxide (1 M) (Bio Basic Canada Inc., Markham, ON, Canada). The mixture was incubated at 37°C for 2 h further.
Lim E.S., Lim M.J., Min K.S., Kwon Y.S., Hwang Y.C., Yu M.K., Hong C.U, & Lee K.W. (2016). Effects of epicatechin, a crosslinking agent, on human dental pulp cells cultured in collagen scaffolds. Journal of Applied Oral Science, 24(1), 76-84.
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6

Isolation and Culture of Murine Tracheal Epithelial Cells

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The protocol was performed as described previously [19] (link). Briefly, at the time of necropsy, the chest and cervical region were exposed. A small puncture was placed in the proximal trachea to allow cannulation with sterile 0.86 mm polyethylene tubing (Intramedic Clay Adams) which was secured in place with a 3.0 silk suture. A loose suture was placed at the distal end of the trachea just proximal to the carina. The trachea was dissected free and immediately placed in DMEM at 4°C. And each trachea gently inflated with 0.2% protease through the tracheal cannula after tightening the distal suture. Dissociated epithelial cells were gently harvested by injecting 5 milliliters of cell culture media through the trachea. MTE cells from all tracheas were pooled and re-suspended in cell culture media prior to plating on Transwell© chambers (Corning) coated with Purcol© (Advanced Biomatrix). MTE cells were maintained in submerged conditions until confluent at which time they were kept in air-liquid interface conditions for one week in the presence of 100 nM retinoic acid. For the experiments, MTE cells were treated with 50 ng recombinant murine IFN γ (R and D systems) and/or 25 µg dsRNA for 24 hours.
Oslund K.L., Zhou X., Lee B., Zhu L., Duong T., Shih R., Baumgarth N., Hung L.Y., Wu R, & Chen Y. (2014). Synergistic Up-Regulation of CXCL10 by Virus and IFN γ in Human Airway Epithelial Cells. PLoS ONE, 9(7), e100978.
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7

Establishing Pancreatic, Liver, and Lung Tumor Cell Lines

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Cell lines were generated from tumor-bearing pancreata, livers, or lungs of KC-shSmad4 or KC-shRen mice. Liver- and lung-derived lines were only used for sWGS analysis. Tumors were dissected, chopped with razor blades, and digested with 1 mg/ml collagenase V (Sigma-Aldrich) diluted in HBSS for 30–60 min, followed by 0.25% trypsin for 5–10 min. Digested tissues were washed with complete DMEM (DMEM, 10% FBS (Gibco), 1X penicillin–streptomycin), passed through a 100-μm filter, and cultured in complete DMEM on collagen-coated plates (PurCol, Advanced Biomatrix, 0.1 mg/ml) supplemented with 1 μg/ml doxycycline at 37°C. Cells were passaged at least five times to eliminate any non-tumor cells before using them in experiments. Primary cultures were authenticated by flow cytometry of engineered fluorescent alleles. All cultures were tested negative for mycoplasma.
Tsanov K.M., Barriga F.M., Ho Y.J., Alonso-Curbelo D., Livshits G., Koche R.P., Baslan T., Simon J., Tian S., Wuest A.N., Luan W., Wilkinson J.E., Masilionis I., Dimitrova N., Iacobuzio-Donahue C.A., Chaligné R., Pe’er D., Massagué J, & Lowe S.W. (2024). Metastatic site influences driver gene function in pancreatic cancer. bioRxiv.
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8

3D Collagen Gel Contraction Assay

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Control, Myocd-LSD1, and Myocd-LSD1NF SMC were resuspended in a 10% FBS F12:DMEM media at the density 5x105 cells/mL after starvation in serum-free, insulin-free media for 48 to 72 hours. The cell suspension was incorporated in a collagen solution [bovine collagen I solution (PurCol®, Advanced Biomatrix, #5005), 10 mM NaOH, 1X PBS] and the cell-collagen solution was deposed on non-treated tissue culture 12-well plates. Plates were incubated at 37°C for 2 to 3 hours to allow gelation. Then, gels were immersed in 10% FBS culture medium and lifted from the bottom of the plate. Gel images were acquired immediately (baseline) and every 24 hours under a dissection microscope. Gel diameter and area were calculated using Image Pro Premier software. Gels were stained with ACTA2-FITC and DAPI (overnight incubation) after Triton-X-100 permeabilization and blocking in 5% BSA 10% horse serum PBS. Cell density was evaluated by counting the number of DAPI+ cells in 3 individual fields/gel and normalized to the field area (cells/mm2). Estimation of the cell number per gel was done as follow: number of cells = Area x Cell density.
Liu M., Espinosa-Diez C., Mahan S., Du M., Nguyen A.T., Hahn S., Chakraborty R., Straub A.C., Martin K.A., Owens G.K, & Gomez D. (2021). H3K4 di-methylation governs smooth muscle lineage identity and promotes vascular homeostasis by restraining plasticity. Developmental cell, 56(19), 2765-2782.e10.
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9

Quantifying Collagenase Activity in Wound Samples

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Wound and explant samples were placed in the upper chambers of transwells in 50 µl, and 1% (v/v) quenched fluorescein-conjugated DQ™ type I collagen (1 µg/ml; Life Technologies, Grand Island, NY) was added to the bottom chambers in 500 µl of phenol red-free RPMI with 1% FBS and 1% penicillin/streptavidin. Collagenase activity was defined as the fluorescence detected in the bottom chamber (excitation 480 nm, emission 530 nm) minus fluorescence in blank controls (no sample in the upper chamber). For 3D matrix, we mixed native type I collagen (PurCol, Advanced BioMatrix, Carlsbad, CA) with DQ collagen and seeded explants onto the mixed matrix and assessed collagenase activity. For antibody depletion, we cultured samples with antibodies against MMP-8 (R&D Systems, Minneapolis, MN) or MMP-13 (Thermos Fisher Scientific, Waltham, MA). For macrophages, cells were incubated with phenol red-free RPMI containing 1% DQ collagen and 6 h.
Rohani M.G., McMahan R.S., Razumova M.V., Hertz A.L., Cieslewicz M., Pun S.H., Regnier M., Wang Y., Birkland T.P, & Parks W.C. (2015). MMP-10 Regulates Collagenolytic Activity of Alternatively Activated Resident Macrophages. The Journal of investigative dermatology, 135(10), 2377-2384.
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10

VEGFR-3 and Syndecan Interactions in Lymphangiogenesis

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Chamber slides (Lab-Tek) coated with 50-μg/mL PurCol (Advanced Biomatrix), layered with serum-starved hLEC were stimulated with human mature VEGF-C (R&D) for 5 minutes ± pre treatment with heparinase in some experiments. Cells were fixed in ice-cold methanol for 10 minutes and blocked (Olink PLA-blocking reagent; 30 minutes at 37°C). Rabbit antihuman VEGFR-3 (Reliatech; or for some studies, anti–VEGFR-2; Cell Signaling) and goat antihuman Sdc-4 (R&D) were then added (2 µg/mL) together (in blocking solution overnight; 4°C). After wash, Rabbit(−) and Goat(+) PLA Probes (Duolink assay; Olink Bioscience) were added (1:10 dilution). In other experiments, rabbit anti–VEGFR-3 was paired with either mouse antihuman Sdc-1 (Abcam) or mouse antihuman Sdc-2 (kind gift from G. David) antibodies. Antibodies were directed against extracellular domains, as per manufacturer protocol (imaging: 40× objective, room temperature).
Johns S.C., Yin X., Jeltsch M., Bishop J.R., Schuksz M., El Ghazal R., Wilcox-Adelman S.A., Alitalo K, & Fuster M.M. (2016). Functional Importance of a Proteoglycan Coreceptor in Pathologic Lymphangiogenesis. Circulation Research, 119(2), 210-221.
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