Collagen 4 coated plates

Manufactured by Corning

Sourced in Switzerland

Corning Collagen IV-coated plates are designed for cell culture applications. The plates are pre-coated with a thin layer of collagen IV, a key structural component of the extracellular matrix. This coating provides a natural substrate for cell attachment and growth.

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Collagen-coated plates (19 citations) Collagen IV (24 citations)

6 protocols using collagen 4 coated plates

Isolation and Culture of Pancreatic Ductal Cells

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The following was performed as previously described^{53 (link)} with modifications. Pancreata were harvested and digested at 37 °C for 45 min (collagenase IV, 4 mg ml⁻¹) and passed through a 100-μm nylon cell strainer to separate the acinar fraction from larger ducts. The ductal fraction underwent additional digestion with 0.25% trypsin (Gibco) for 5 min at 37 °C and mechanical disruption. The two fractions were combined and plated on collagen IV-coated plates (Corning) in modified PDEC medium: DMEM/F12 (Gibco), 15 mM HEPES (Invitrogen), 5 mg ml⁻¹d-glucose (Sigma Aldrich), 1.22 mg ml⁻¹ nicotinamide (Sigma Aldrich), 5 nM 3,3,5-tri-iodo-l-thyronine (Sigma Aldrich), 1 μM dexamethasone (Sigma Aldrich), 100 ng ml⁻¹ cholera toxin (Sigma Aldrich), 5 ml l⁻¹ insulin-transferrin-selenium (BD), penicillin (Gibco), 100 μg ml⁻¹ streptomycin (Gibco), 0.1 mg ml⁻¹ soybean trypsin inhibitor (Sigma Aldrich), 40 ng ml⁻¹ EGF (Sigma Aldrich), 25 μg ml⁻¹ bovine pituitary extract (Invitrogen), 100 μM β-mercaptoethanol (Sigma) and 10% FBS (Gibco). Cells were passaged at low confluency until exhaustion or escaper clones were established.

Genovese G., Carugo A., Tepper J., Robinson F.S., Li L., Svelto M., Nezi L., Corti D., Minelli R., Pettazzoni P., Gutschner T., Wu C.C., Seth S., Akdemir K.C., Leo E., Amin S., Molin M.D., Ying H., Kwong L.N., Colla S., Takahashi K., Ghosh P., Giuliani V., Muller F., Dey P., Jiang S., Garvey J., Liu C.G., Zhang J., Heffernan T.P., Toniatti C., Fleming J.B., Goggins M.G., Wood L.D., Sgambato A., Agaimy A., Maitra A., Roberts C.W., Wang H., Viale A., DePinho R.A., Draetta G.F, & Chin L. (2017). Synthetic vulnerabilities of mesenchymal subpopulations in pancreatic cancer. Nature, 542(7641), 362-366.

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Neuroblast Differentiation and Lentiviral Transduction

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Undifferentiated PC12 cells were plated onto collagen IV-coated plates (Corning) and differentiated in culture medium containing 50 ng/ml NGF (Accurate Chemical) for 5 – 7 days. Primary sympathetic neuroblasts isolated from SCGs of post-gestation rat pups (P2) were cultured as previously described [20 (link)]. Differentiated PC12 cells and neuroblasts were infected with lentivirus encoding shXAF1 or nontargeting control virus (shSCR) followed by NGF withdrawal with anti-NGF antibody (1:5000, Sigma) for up to 48 hours where they were harvested for subsequent analysis.

Choo Z., Lin Koh R.Y., Wallis K., Wei Koh T.J., Kuick C.H., Sobrado V., Kenchappa R.S., Pheng Loh A.H., Soh S.Y., Schlisio S., En Chang K.T, & Chen Z.X. (2016). XAF1 promotes neuroblastoma tumor suppression and is required for KIF1Bβ-mediated apoptosis. Oncotarget, 7(23), 34229-34239.

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Silencing ATF3 and PBK in Corneal Epithelial Cells

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hTCEpi cells were cultured in keratinocyte–serum-free media (K-SFM) with supplements (Invitrogen). Primary HCEKs and HLEKs were isolated from cadaver donor corneas provided by Midwest Eye Bank (Ann Arbor, MI, USA) and cultured in CnT-20 media with supplements (CellnTech, Bern, Switzerland) on collagen IV-coated plates (Corning, Corning, NY, USA). To silence gene expression, small interfering RNAs (siRNAs) (20 nM) targeting ATF3 (Catalog #AM16708; Invitrogen), PBK (Catalog #4390824; siPBK#1: Assay ID s31708, siPBK#2: Assay ID s31707, siPBK#3: Assay ID s31706; Invitrogen) or a scrambled negative control (Catalog #465372; Invitrogen) were transiently transfected into cells using RNAiMAX (Invitrogen). Although we have tested three siRNA sequences for PBK1, only two of them (#1 and #3) reduced the expression efficiently.

Kaplan N., Wang J., Wray B., Patel P., Yang W., Peng H, & Lavker R.M. (2019). Single-Cell RNA Transcriptome Helps Define the Limbal/Corneal Epithelial Stem/Early Transit Amplifying Cells and How Autophagy Affects This Population. Investigative Ophthalmology & Visual Science, 60(10), 3570-3583.

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Differentiation and Oxidative Stress Analysis of PC12 Cells

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Undifferentiated PC12 cells were plated onto collagen IV-coated plates (Corning) and differentiated in culture medium containing 50 ng/ml NGF (Accurate chemicals) for 5–7 days as described^{20 (link),21 (link)}. Differentiated PC12 were pre-treated with 10 mM N-acetylcysteine (NAC) (Sigma Aldrich) before NGF withdrawal and continuously replenished every 12 hours after NGF withdrawal with anti-NGF antibody (1:5000, Sigma Aldrich) for up to 48 hours where they were harvested for Immunoblot analysis.

Angelina C., Tan I.S., Choo Z., Lee O.Z., Pervaiz S, & Chen Z.X. (2017). KIF1Bβ increases ROS to mediate apoptosis and reinforces its protein expression through O2− in a positive feedback mechanism in neuroblastoma. Scientific Reports, 7, 16867.

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Evaluating Cell Adhesion on ECM Coatings

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In total, 96-well NUNC plates were coated with different ECM for two days at 4 °C; 25 μg ml⁻¹ Collagen I (Corning, VWR, Vienna, Austria, cat#354236) (in 0.02 N acetic acid), 25 μg ml⁻¹ Fibronectin (Gibco, cat#PHE0023) (in PBS) or 10 μg ml⁻¹ Laminin V (BioLamina, cat#LN111-02) (in PBS with Ca²⁺/Mg²⁺). Unbound Collagen I and Fibronectin were removed with PBS and excess Laminin V with PBS with Ca²⁺/Mg²⁺. Wells were then blocked with 0.5% heat-inactivated bovine serum albumin (BSA) in PBS (for Fibronectin and collagens) or in PBS with Ca²⁺/Mg²⁺ (for Laminin V) and incubated overnight at 4 °C. Totally, 2 × 10⁴ cells were seeded in to each plate including commercial Collagen IV-coated plates (Corning, cat#08-774-30) and incubated at 15, 30, 45, 60, and 100 min at 37 °C before washing 3 times with PBS. Cells were then incubated with CnT-PR media before applying 15 μM of Resazurin for 75 min. Fluorescence was read at 560ex/590em, sensitivity 40 (Synergy HT from Biotek + Biostack and Gen5 software).

Mercado N., Schutzius G., Kolter C., Estoppey D., Bergling S., Roma G., Gubser Keller C., Nigsch F., Salathe A., Terranova R., Reece-Hoyes J., Alford J., Russ C., Knehr J., Hoepfner D., Aebi A., Ruffner H., Beck T.C., Jagannathan S., Olson C.M., Sheppard H.E., Elsarrag S.Z., Bouwmeester T., Frederiksen M., Lohmann F., Lin C.Y, & Kirkland S. (2019). IRF2 is a master regulator of human keratinocyte stem cell fate. Nature Communications, 10, 4676.

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Isolation and Cultivation of Pancreatic Tumor Cells

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PDAC-PDXs were generated accordingly to Kim MP et al (17 (link)). Early passage PDXs (F1) from primary human pancreatic ductal adenocarcinoma were harvested in cold HBSS (Gibco) and tumor cells isolated through mechanical and enzymatic dissociation. Briefly, tumors were minced in very small pieces with scissors under sterile conditions before being processed with the Human Tumor Dissociation Kit (Miltenyi Biotec). After digestion, single isolated cells were seeded at high confluency on collagen-IV-coated plates (Corning) in DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% BSA (Fisher Scientific), 0.5 μM hydrocortisone (Sigma-Aldrich), 10 mM HEPES (Invitrogen), 100 ng/ml cholera toxin (Sigma-Aldrich), 5 ml/l insulin-transferrin-selenium (BD), 100 IU/ml penicillin (Gibco), and 100 μg/ml streptomycin (Gibco). To remove fibroblasts in the culture, we periodically performed brief trypsinization (0.25% Trypsin-EDTA, Gibco). The purity of the human culture derived from PDX was confirmed over time by flow cytometry through the evaluation of HLA-ABC and mouse H-2Kd histocompatibility complex antigens (Becton Dickinson). Isolated human cells were maintained in culture for a maximum of couple of passages before being switch to DMEM 10% FBS and enrolled in phenotypic studies and molecular profiling.

Tomihara H., Carbone F., Perelli L., Huang J.K., Soeung M., Rose J.L., Robinson F.S., Deribe Y.L., Feng N., Takeda M., Inoue A., Del Poggetto E., Deem A.K., Maitra A., Msaouel P., Tannir N.M., Draetta G.F., Viale A., Viale A., Heffernan T.P., Bristow C.A., Carugo A, & Genovese G. (2020). Loss of ARID1A promotes epithelial-mesenchymal transition and sensitizes pancreatic tumors to proteotoxic stress. Cancer research, 81(2), 332-343.

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