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CBA013 InnoCyte™ ECM Cell Adhesion Assay, Collagen Type IV

CBA013
  
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Fluorometric
      Description
      OverviewA convenient assay for the determination of the relative attachment of adherent cell lines to collagen type IV. Cells are seeded onto the collagen type IV coated plates followed by determination of relative cell attachment using a fluorescent dye.
      Catalogue NumberCBA013
      Brand Family Calbiochem®
      Materials Required but Not Delivered Pipettors with disposable tips
      Fluorescence reader capable of measuring fluorescence in 96-well plates with a filter set of excitation wavelength ~485 nm and emission wavelength ~520 nm
      PBS, preferably containing calcium/magnesium (Dulbecco's PBS) for washing
      References
      ReferencesCaroll, D.K., et al. 2006. Nat. Cell Biol. 8, 551.
      Hynes, R.O., et al. 1999. Proc. Natl. Acad. Sci. USA96, 2588.
      Ohashi, T., et al. 1999. Proc. Natl. Acad. Sci USA 96, 2153.
      Grande, J.P., et al. 1997. J. Lab. Clin. Med. 130, 476.
      Nelson, P.R., et al. 1997. Vasc. Surg. 26, 104.
      Horowitz, A.F., et al. 1996. Trends Cell Biol. 6, 460.
      Hynes, R.O., et al. 1990. Fibronectins, Springer-Verlag, New York.
      Preissner, K.T., et al. 1990. J. Biol. Chem. 265, 18490.
      Product Information
      Detection methodFluorometric
      Form72 Tests
      Format96-well plate
      Kit containsCoated 96-Well Plate, Fluorescent Dye, D-PBS, and a user protocol.
      Applications
      Application ReferencesCaroll, D.K., et al. 2006. Nat. Cell Biol. 8, 551.
      Biological Information
      Assay time2.5 h
      Sample TypeAdherent cultured cells
      Physicochemical Information
      Emission max.
      Excitation max.
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Innocyte™ Cell Adhesion Microplate Assays are designed for the determination of the relative attachment of adherent cell lines to extracellular matrix proteins such as Human Fibronectin (Cat. No. CBA011), Human Vitronectin (Cat. No. CBA012), and Human Collagen IV (Cat. No. CBA013). Cells are seeded onto a coated substrate, which is followed by the determination of relative cell attachment using a fluorescent dye.
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage Multiple storage requirements
      Storage ConditionsUpon arrival store the Calcein-AM at -20°C and the remaining components at 4°C.
      Protect from Light Protect from light
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsCoated 96-Well Plate, Fluorescent Dye, D-PBS, and a user protocol.
      Specifications

      Documentation

      InnoCyte™ ECM Cell Adhesion Assay, Collagen Type IV Certificates of Analysis

      TitleLot Number
      CBA013

      References

      Reference overview
      Caroll, D.K., et al. 2006. Nat. Cell Biol. 8, 551.
      Hynes, R.O., et al. 1999. Proc. Natl. Acad. Sci. USA96, 2588.
      Ohashi, T., et al. 1999. Proc. Natl. Acad. Sci USA 96, 2153.
      Grande, J.P., et al. 1997. J. Lab. Clin. Med. 130, 476.
      Nelson, P.R., et al. 1997. Vasc. Surg. 26, 104.
      Horowitz, A.F., et al. 1996. Trends Cell Biol. 6, 460.
      Hynes, R.O., et al. 1990. Fibronectins, Springer-Verlag, New York.
      Preissner, K.T., et al. 1990. J. Biol. Chem. 265, 18490.
      User Protocol

      Revision09-June-2008 JSW
      Form72 Tests
      Format96-well plate
      Detection methodFluorometric
      StorageUpon arrival store the Calcein-AM at -20°C and the remaining components at 4°C.
      Intended useThe Innocyte™ Cell Adhesion Microplate Assays are designed for the determination of the relative attachment of adherent cell lines to extracellular matrix proteins such as Human Fibronectin (Cat. No. CBA011), Human Vitronectin (Cat. No. CBA012), and Human Collagen IV (Cat. No. CBA013). Cells are seeded onto a coated substrate, which is followed by the determination of relative cell attachment using a fluorescent dye.
      BackgroundThe extracellular matrix (ECM) provides vital structure and organization to most organisms. It is an assemblage of collagens, proteoglycans, and glycoprotein such as fibronectin and vitronectin. The ECM undergoes a constant remodeling that is most obvious during development, wound healing, and other repair processes. Fibronectin is a prominent constituent of the ECM around many cells, and fibronectin-rich matrices provide substrates for cell adhesion and migration during development, wound healing, and other processes, and affect many cellular functions, including proliferation, survival, and differentiation. Vitronectin is a member of the pexin family. It is found in serum and tissues and promotes cell adhesion and spreading, inhibits the membrane-damaging effect of the terminal cytolytic complement pathway, and binds to several serpin serine protease inhibitors. Collagen type IV is a prominent component of the basement membrane. Collagen IV can stimulate tumor cell proliferation, migration, and adhesion in a dose-dependent manner. Powerful cytokines such as TGF-β1 play an important role in stimulating the expression of collagen IV. Adhesive receptors on the cell surface such as integrins, selectins, cadherins, and ICAMs interact in a specific manner with different ECM proteins. Adhesion molecules reside in complex structures like focal adhesion adhesions and synaptic and adherens junctions, which also contain many specialized cytoskeletal proteins and signaling molecules.
      Principles of the assayThe Innocyte™ Cell Adhesion Assay is designed for the analysis of cell attachment to ECM proteins. After incubation followed by a brief wash step, attached cells are quantified with the green fluorescent dye calcein-AM. BSA-coated wells serve as a negative control, and poly-L-lysine-coated wells serve as a positive control for general attachment.
      Materials provided• Collagen Type IV-Coated 96-Well Plate (Kit Component No. JA7860-1EA): 1 black plate, 96 wells, supplied as six 16-well strips; Rows 2-7 are coated with collagen type IV, row 1 is coated with BSA, and row 8 is coated with poly-L-lysine.
      • Calcein-AM Solution (Kit Component No. JA7705-50UL): 1 amber vial, 50 µl
      • D-PBS (Kit Component No. JA7706-10ML): 1 plastic bottle, 10 ml
      Materials Required but not provided Pipettors with disposable tips
      Fluorescence reader capable of measuring fluorescence in 96-well plates with a filter set of excitation wavelength ~485 nm and emission wavelength ~520 nm
      PBS, preferably containing calcium/magnesium (Dulbecco's PBS) for washing
      Reagent preparationPrepare the calcein-AM working solution as follows. Warm the calcein-AM solution and the D-PBS to room temperature. Dilute the calcein-AM stock solution 1:300 with D-PBS, i.e. add 20 µl (calcein-AM solution) to 6 ml of D-PBS. This calcein-AM working solution cannot be stored.
      Detailed protocol1. Harvest cells and resuspend the cell pellet in serum-free media. Cell density should be between 100,000 and 500,000 cells/ml.
      2. Add 100 µl of the prepared cells suspension, in duplicate, to the wells and incubate for 1-2 h at 37°C in a cell culture incubator.
      3. Discard the cell suspension and gently wash wells twice with 200 µl of PBS.
      4. Add 100 µl of the calcein-AM working solution to each well.
      5. Incubate for 1 h at 37°C in a cell culture incubator.
      Measure the fluorescence of the samples using a fluorescence plate reader at excitation wavelength ~485 nm and emission wavelength ~520 nm.
      Assay characteristics and examples

      Table 1: Assay Template


      Figure 1: Example Data

      ~25,000 cells were added to each well and allowed to attach for 1.5 hours at 37°C in 6% COS2. Cells were washed gently with D-PBS and labeled with calcein-AM for 1 h at 37°C in 6% CO2. The relative attachment of cells to poly-L-lysine was determined for HUVECS only and was lower than that of the displayed ECM proteins (data not shown).

      Application referencesCaroll, D.K., et al. 2006. Nat. Cell Biol. 8, 551.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      InnoCyte™ and InteractivePathways™ are trademarks of EMD Chemicals, Inc.