Collagen 2

Manufactured by Merck Group

Sourced in United States

Collagen II is a type of collagen protein that is a major structural component of cartilage. It is used in various laboratory applications for research and testing purposes.

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Lab products found in correlation

Fibrinogen, by Enzyme Research (3 mentions) Fibronectin, by Enzyme Research (3 mentions) Fibronectin, by Merck Group (2 mentions) Collagen 4, by Merck Group (2 mentions) Collagen 1, by Merck Group (2 mentions) Lsm 710, by Zeiss (2 mentions) Goat anti rabbit secondary antibody, by Proteintech (1 mentions) Collagenase type 2, by Merck Group (1 mentions) Dmem f12, by Thermo Fisher Scientific (1 mentions) Ici 182 780, by Merck Group (1 mentions) Trypsin, by Merck Group (1 mentions) Annexin 5 fitc pi kit, by BD (1 mentions) Endothelial cell medium (ecm), by ScienCell (1 mentions) Activated caspase 3, by Bioworld Technology (1 mentions) Collagen x, by Abcam (1 mentions) Dab horseradish peroxidase color development kit, by ZSGB-BIO (1 mentions) Formaldehyde, by Ted Pella (1 mentions) Laminin, by Merck Group (1 mentions) Cell culture reagents, by Thermo Fisher Scientific (1 mentions) Octadecanethiol, by Merck Group (1 mentions)

12 protocols using collagen 2

Chondrocyte Isolation and Culture

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We used the following reagents: DMEM/F12 (Gibco, USA), fetal bovine serum (FBS) (BI, Israel), trypsin (Sigma, USA), collagenase type II (Sigma, USA), D-Hanks and PBS (Solarbio, Beijing, China), Annexin V-FITC/PI kit (BD, USA), primary antibody of anti-α1 (Proteintech, Wuhan, China), E2 (Sigma, USA), collagen II (Sigma, USA), ICI182780 (Sigma, United Kingdom), and secondary antibody (goat anti-rabbit) (Proteintech, Wuhan, China).

Zhao C.M., Chen Q., Zhang W.J., Huang A.B., Zhang W., Yang H.L, & Zhang Z.M. (2016). 17β-Estradiol Protects Rat Annulus Fibrosus Cells Against Apoptosis via α1 Integrin-Mediated Adhesion to Type I Collagen: An In-vitro Study. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 22, 1375-1383.

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Culturing Primary Human Umbilical Vein Endothelial Cells

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Primary human umbilical
vein endothelial cells (HUVECs) were isolated from the neonatal umbilical
cord (Dalian Obstetrics and Gynecology Hospital) and cultured in an
endothelial cell medium (ECM, ScienCell) supplemented with 10% fetal
bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C
with 5% CO₂. The MCF7 cell line was also cultured in the
ECM under the same culture conditions. Cells were resuspended at a
density of 1.0 × 10⁶ cells/mL and then seeded on collagen
II (2 μg/cm², Sigma-Aldrich) pretreated microwells.
Thereafter, HUVECs were stained by MitoGreen (1/1000 dilution, #KGMP0072,
KeyGEN BioTECH) at 37 °C for 30 min, and then, cell suspension
(15 μL) was injected into the microvascular model. Then, the
chip was incubated at 37 °C with 5% CO2, and the cultural medium
in the chip was replaced every 8 h until the cells in the chip reached
80% confluence.

Hao Z., Lv H., Tan R., Yang X., Liu Y, & Xia Y.L. (2021). A Three-Dimensional Microfluidic Device for Monitoring Cancer and Chemotherapy-Associated Platelet Activation. ACS Omega, 6(4), 3164-3172.

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Assessing Cartilage Development in Growth Plates

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Limbs were fixed in 4% paraformaldehyde for 24 h and decalcified in 10% EDTA for 3 days. Paraffin sections (4-µm) were obtained and stained with Hematoxylin and Eosin (H&E), safranin O and toluidine blue. Mean values of heights of the reserve, proliferative, hypertrophic zones, and the total growth plate were calculated from measurements taken at 3 positions across the proximal tibia growth plates using ImageJ version 1.44p software. IHC/Immunocytochemistry (ICC) was performed with Hsitostain-Plus kit (ZSGB-BIO, China). Primary antibodies included: collagen II (Sigma, USA), sox 9 (Abcam, UK), collagen X (Abcam, UK), and activated-caspase 3 (Bioworld, USA). Detection was conducted with a DAB horseradish peroxidase color development kit (ZSGB-BIO, China). Semi quantitative analysis of IHC images through integrated optical density (IOD) was taken using Image-Pro Plus version 6.0.0.260 software.

Liang G., Lian C., Huang D., Gao W., Liang A., Peng Y., Ye W., Wu Z., Su P, & Huang D. (2014). Endoplasmic Reticulum Stress-Unfolding Protein Response-Apoptosis Cascade Causes Chondrodysplasia in a col2a1 p.Gly1170Ser Mutated Mouse Model. PLoS ONE, 9(1), e86894.

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Cell Adhesion Protein Sourcing

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General laboratory chemicals and reagents were purchased from VWR. Cell culture reagents, and antibodies were purchased from Life Technologies. Octadecanethiol, fibronectin, collagen IV, collagen II, and laminin were purchased from Sigma-Aldrich. Formaldehyde was purchased from Ted Pella, Inc.

Sobers C.J., Wood S.E, & Mrksich M. (2015). A Gene Expression-Based Comparison of Cell Adhesion to Extracellular Matrix and RGD-Terminated Monolayers. Biomaterials, 52, 385-394.

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Measuring IgM Reactivity in RA Patients

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To analyze the reactivity of CD27⁺IgD⁺ B cells-derived IgM, microtiter plates were coated with the well-studied RA autoantigens, including ssDNA, fibrinogen, vimentin, and collagen II (Sigma, 10 µg/ml each). BSA was chosen as the control. 100 µl culture supernatants of the CD27⁺IgD⁺ B cells (cultured as described above) from both healthy individuals and RA patients were tested, with the medium only as the background control. Biotin-conjugated goat anti-human IgM and HRP-labeled streptavidin (BETHYL, Montgomery, TX, USA) were used for the IgM detection, with TMB (Neobioscience Technology, Beijing, China) as the substrate. OD450 was measured using a microplate reader (Bio-Tek, Winooski, VT, USA). The reactivity was demonstrated as (OD450_{cell culture supernatants} − OD450_{medium control}).

Hu F., Zhang W., Shi L., Liu X., Jia Y., Xu L., Zhu H., Li Y., Xu D., Lu L., Qiu X., Liu W., Qiao J., Wang Y, & Li Z. (2018). Impaired CD27+IgD+ B Cells With Altered Gene Signature in Rheumatoid Arthritis. Frontiers in Immunology, 9, 626.

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Isolation and Inhibition of Chondrocytes

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We isolated chondrocytes from the knee joints of two-week-old Wistar rats. Bilateral knee cartilage tissues were extracted and cut up using scissors in a sterile environment. The cartilage was bathed in 0.1% trypsin (Beyotime, Shanghai, China) for 60 min, followed by overnight incubation in 0.2% collagen II (Sigma, St. Louis, MO, USA) in a cell culture incubator. The digested cells were collected and resuspended in chondrocyte medium (DMEM/F-12) for culturing (BI, Kibbutz Beit Haemek, Israel). The chondrocytes in the medium were then placed in a cell culture incubator for passaging. Subsequent experiments were performed using second-generation chondrocytes. To inhibit the SIRT1/FOXO1 signaling pathway, serum-starved cartilage cells were pretreated with SIRT1 inhibitors EX-527 (10 μM/mL) or FOXO1 inhibitors AS (1 μM/mL) for 6 h and then exposed to IL-1β (10 ng/mL) in the presence or absence of resveratrol (50 μM) for 24 h.

Liang C., Xing H., Wang C., Xu X., Hao Y, & Qiu B. (2022). Resveratrol protection against IL-1β-induced chondrocyte damage via the SIRT1/FOXO1 signaling pathway. Journal of Orthopaedic Surgery and Research, 17, 406.

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Scaffold-Seeded MSC Immunohistochemistry

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MSCs seeded on the scaffolds (n = 6) were cultured and harvested at the end of 1 and 2 weeks. The samples were washed with PBS and fixed in 10% neutral buffered formalin. Thereafter, they were dehydrated through a series of graded alcohols and embedded in paraffin. Sections of 5 μm thickness were cut and collected on slides. For immunohistochemistry stains, the slides were incubated with collagen II (Sigma Co.) and TGF-β3 (Sigma Co.) antibodies. Then detection was applied using streptavidin-biotin immunoenzymatic antigen detection system (UltraVision Detection System, LabVision, USA). The results were assessed by three individuals who were blinded to the treatment.

Sun L., Li H., Qu L., Zhu R., Fan X., Xue Y., Xie Z, & Fan H. (2014). Immobilized Lentivirus Vector on Chondroitin Sulfate-Hyaluronate Acid-Silk Fibroin Hybrid Scaffold for Tissue-Engineered Ligament-Bone Junction. BioMed Research International, 2014, 816979.

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Collagen-induced RA fibroblasts treated with PG

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The human RA synovial fibroblasts MH7A was purchased from Riken (Saitama, Japan) and grew in Roswell Park Memorial Institute (RPMI) 1640 (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA), 100 µg/mL streptomycin and 100 U/mL penicillin (Invitrogen, Carlsbad, CA, USA). Lyophilized bovine type II collagen (C II) powder was dissolved in acetic acid (0.05 M) at a concentration of 2.0 mg/mL. Then, cells were induced by Collagen II (C II, Sigma-Aldrich Co. St. Louis, MO, USA) for 24 h. PG (purity >98%) were purchased from Nantong Feiyu Biological Technology Co., Ltd. (Nantong, Jiangsu, China), which chemical formula was illustrated in Figure 1A. The stock solution of PG (1 mg/mL) was diluted with RPMI 1640 until the final concentrations were 10, 20, and 50 µg/mL, respectively (22 (link)). The MH7A cells were grouped as follows: the control group: MH7A cells without any treatment; collagen induced RA model group (RA); RA + PG (10, 20, 50 µg/mL): collagen induced RA MH7A cells were treated by 10, 20, and 50 µg/mL PG, respectively for 24 h at 37 °C in an atmosphere of 5% CO₂.

Fu M., Sang X, & Cheng H. (2022). Total glucosides of peony induce fibroblast-like synovial apoptosis, and ameliorate cartilage injury via blocking the NF-κB/STAT3 pathway. Annals of Translational Medicine, 10(2), 51.

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Integrin-α10 Co-Immunoprecipitation and Signaling

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For the co-immunoprecipitation and collagen II-induced signaling experiments, MXF8000 cells were detached using enzyme-free detachment medium (Gibco) and kept in suspension in serum-free medium with 2% BSA for 30 min, then replated on dishes pre-coated with collagen II (Millipore) at 10 μg/ml. Cells were harvested three hours later (for immunoprecipitations) or at the times indicated in the figure. For immunoprecipitations, total lysates were prepared in lysis buffer (20 mM Tris-HCL, 150 mM NaCl, 1% Triton-X100, 5 mM MgCl₂, 1 mM EDTA, proteinase inhibitor cocktail [SIGMA], and phosSTOP tablet [Roche]). Lysate was subjected to immunoprecipitation using anti-integrin-α10 (AB6030) or rabbit IgG (SIGMA), then immunocomplexes were subjected to Western blotting using anti-integrin-α10 monoclonal antibody (Neobio).
GTP-RAC was assessed using a GST-PBD pulldown assay as described (60 (link)).

Okada T., Lee A.Y., Qin L.X., Agaram N., Mimae T., Shen Y., O’Connor R., López-Lago M.A., Craig A., Miller M.L., Agius P., Molinelli E., Socci N.D., Crago A.M., Shima F., Sander C, & Singer S. (2016). Integrin-α10 dependency identifies RAC and RICTOR as therapeutic targets in high-grade myxofibrosarcoma. Cancer discovery, 6(10), 1148-1165.

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Primary Myxofibrosarcoma Cell Lines

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Following guidelines in IRB-approved protocol 02-060, primary myxofibrosarcoma cell lines (MXF8500, MXF2734, MXF8000, and MXF9100) were derived during 2004 to 2011 from fresh human primary myxofibrosarcoma tumor samples using collagenase digestion and established in cell culture. Array CGH was performed on all primary cell lines and compared to array CGH performed on the human tumor tissue from which they were derived so as to verify that the copy number alterations in the cell lines were representative of those found in the original tumor samples. The cell lines were last tested in 2014.
Adipose-derived stem cells (ASCs) were a generous gift from Dr. Jeffrey M. Gimble, and SGBS cells were a generous gift from Dr. Martin Wabitsch. Umbilical cord-derived human mesenchymal stem cells and human dermal fibroblast cells (KEL-FIB) were from ATCC. All cells were grown in a 50:50 mixture of Dulbecco’s modified Eagle’s medium (DMEM) high glucose and F12 medium (DMEM HG/F12) with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin and maintained in a 37°C incubator with 5% CO₂. Collagen I was from Sigma and Collagen II from Millipore. NSC23766 and INK128 were obtained from Cayman, EHop-016 from Millipore and Selleckchem, IPA3 from Tocris.

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