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CBA025 InnoCyte™ ECM Cell Adhesion Assay, Laminin/Basement Membrane Complex (BMC)

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CBA025
  
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Fluorometric
      Description
      Overview

      This product has been discontinued.





      The InnoCyte™ Cell Adhesion Assay, Laminin I/Basement Membrane Complex is designed for the determination of the relative attachment of adherent cell lines to laminin I and basement membrane protein complex, for evaluation of cell adhesion receptors, and for assessment of inhibitory or stimulatory substances for cell attachment. The kit is supplied with a 96-well strip plate coated with mouse laminin I and basement membrane complex (see plate format below). Cells are seeded in the coated wells and incubated at +37°C. Following incubation, the wells are washed briefly and attached cells are labeled 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. Relative cell attachment is assessed using a fluorescence plate reader.

      • Convenient: 96-well plate format; allows cell attachment profiling of various cell lines at the same time
      • Sensitive: cell-permeable fluorescent dye; no cell permeabilization needed
      • Easy to use: does not require tedious staining method or expensive complicated equipment

      Catalogue NumberCBA025
      Brand Family Calbiochem®
      Application Data
      ~40,000 cells were added to wells containing laminin I, BMC, or BSA and incubated for 1.5 h at 37°C in a cell culture incubator in the presence of 6% CO2. Cells were washed gently with D-PBS and labeled with Calcein-AM for 1 h at 37°C in a cell culture incubator in the presence of 6% CO2. Fluorescence was measured as indicated in the Detailed Protocol. HT-1080 cells displayed appreciable binding to poly-L-lysine and served as a positive control (data not shown).
      Materials Required but Not Delivered• Cells and cell culture medium
      • Fluorescence reader capable of measuring fluorescence in 96-well plates at an excitation wavelength or ~485 nm and an emission wavelength of ~520 nm
      • 1X PBS for plate washing
      References
      ReferencesCaroll, D.K., et al. 2006. Nat. Cell Biol. 8, 551.
      Ekblom, P., et al. 2003. Matrix Biology 22, 35.
      Li, S., et al. 2002. J. Cell Biol. 157, 1279.
      Hynes, R.O., et al. 1999. Proc. Natl. Acad. Sci. USA 96, 2588.
      Aumailley, M., et al. 1998. J. Anat. 193, 1.
      Nelson, P.R., et al. 1997. Vasc. Surg. 26, 104.
      Horowitz, A.F., et al. 1996. Trends Cell Biol. 6, 460.
      Baron von Evercooren, A., et al. 1982. J. Neurosci. Res. 8, 179.
      Product Information
      Detection methodFluorometric
      Form72 Tests (36 Laminin and 36 BMC)
      Format96-well plate
      Kit containsCoated 96-Well Plates, 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
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe InnoCyte™ Cell Adhesion Assay, Laminin I/Basement Membrane Complex is designed for the determination of the relative attachment of adherent cell lines to laminin I and basement membrane protein complex, for evaluation of cell adhesion receptors, and for screening cell adhesion antagonists.
      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 Plates, Fluorescent Dye, D-PBS, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      CBA025 0

      Documentation

      InnoCyte™ ECM Cell Adhesion Assay, Laminin/Basement Membrane Complex (BMC) Certificates of Analysis

      TitleLot Number
      CBA025

      References

      Reference overview
      Caroll, D.K., et al. 2006. Nat. Cell Biol. 8, 551.
      Ekblom, P., et al. 2003. Matrix Biology 22, 35.
      Li, S., et al. 2002. J. Cell Biol. 157, 1279.
      Hynes, R.O., et al. 1999. Proc. Natl. Acad. Sci. USA 96, 2588.
      Aumailley, M., et al. 1998. J. Anat. 193, 1.
      Nelson, P.R., et al. 1997. Vasc. Surg. 26, 104.
      Horowitz, A.F., et al. 1996. Trends Cell Biol. 6, 460.
      Baron von Evercooren, A., et al. 1982. J. Neurosci. Res. 8, 179.
      User Protocol

      Revision09-June-2008 JSW
      Form72 Tests (36 Laminin and 36 BMC)
      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 Assay, Laminin I/Basement Membrane Complex is designed for the determination of the relative attachment of adherent cell lines to laminin I and basement membrane protein complex, for evaluation of cell adhesion receptors, and for screening cell adhesion antagonists.
      BackgroundThe extracellular matrix (ECM) provides vital structure and organization to most organisms. It is comprised of collagens, proteoglycans, and glycoproteins, such as fibronectin, vitronectin, or laminin. The ECM undergoes a constant remodeling that is most obvious during development, wound healing, and other repair processes. Laminin I, a major component of basement membranes, has numerous biological activities including promotion of cell adhesion, migration, growth, and differentiation, including neurite outgrowth. Laminin I has been shown in culture to stimulate neurite outgrowth, promote cell attachment, chemotaxis, and cell differentiation. It appears early during epithelial morphogenesis in most tissues of the embryo, and remains present as a major epithelial laminin-1 in some adult tissues. Previous organ culture studies with embryonic tissues have suggested that laminin-1 is important for epithelial development. Recent data using genetically manipulated embryonic stem (ES) cells grown as embryoid bodies provide strong support for the view of a specific role of laminin I in epithelial morphogenesis. Adhesive receptors on the cell surface such as integrins, selectins, cadherins, and ICAMs interact in a specific manner with different ECM proteins. The β1 integrins, complexed with various integrin α-subunits, are major adhesion receptors for laminins (for instance α1β1). 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. The Basement Membrane Protein Complex (BMC) is a solubilized basement membrane preparation extracted from EHS (Engelbreth-Holm-Swarm) mouse sarcoma. Its major component is laminin, followed by collagen IV, heparan sulfate proteoglycans, and entactin. It represents a physiologically relevant environment for studies of cell morphology, biochemical function, migration, invasion, and gene expression.
      Principles of the assayThe InnoCyte™ Cell Adhesion Assay, Laminin I/Basement Membrane Complex is designed for the analysis of relative cell attachment to ECM proteins. The kit is supplied with a 96-well strip plate coated with mouse laminin I and basement membrane complex (see plate format below). Cells are seeded in the coated wells and incubated at 37°C. Following incubation, the wells are washed briefly and attached cells are labeled 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. Relative cell attachment is assessed using a fluorescence plate reader.
      Materials provided• Laminin/BMC-Coated 96-Well Plate (Kit Component No. JA7980-1EA): packaged as a single 96-well plate, including frame, pre-coated as follows:
      • Laminin-Coated 96-well Plate Strips: Three 2 x 8-well strips coated with mouse laminin I, BSA, or poly-L-lysine (see configuration below)
      • BMC-Coated 96-well Plate Strips: Three 2 x 8 well strips coated with basement membrane complex (BMC), BSA, or poly-L-lysine (see configuration below)

      Table 1: Sample Plate

      Row A is coated with BSA, rows B-G are coated with laminin I (LM) or BMC, and row H is coated with poly-L-lysine (P-Lys)


      • 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• Cells and cell culture medium
      • Fluorescence reader capable of measuring fluorescence in 96-well plates at an excitation wavelength or ~485 nm and an emission wavelength of ~520 nm
      • 1X PBS for plate washing
      Reagent preparationCalcein-AM Working Solution: prepare immediately prior to use; the Calcein-AM Working Solution cannot be stored for future assays. • Warm the Calcein-AM Solution and the D-PBS to room temperature • Dilute an appropriate amount of Calcein-AM Solution 1:300 with D-PBS For example: to prepare 6 ml Calcein-AM Working Solution add 20 µl Calcein-AM Solution to 5980 µl D-PBS.
      Detailed protocol1. Grow cells of choice in culture medium as appropriate (70-80% confluent).
      2. Harvest the cells by centrifugation at 1000 rpm for 5 min at room temperature. Adherent cells may be trypsinized, followed by centrifugation at 1000 rpm for 5 min at room temperature. Resuspend the cell pellet in serum-free culture medium. The recommended cell density is 100,000 to 500,000 cells/ml.
      3. Remove required number of strips from the Laminin/BMC-Coated 96-Well Plate and place them in the 96-well frame. Return the unused strips to the foil pouch and reseal the entire edge. Store unused strips at 4°C.
      4. Add 100 µl of the cell suspension (10,000-50,000/well) from step 2 to the desired duplicate wells of the Laminin/BMC-Coated 96-Well Plate and incubate for 1-2 h at 37°C in a cell culture incubator.
      5. Discard the cell suspension by shaking the contents of the wells into an approved biological waste container. Gently wash the plate by adding 200 µl 1X PBS to each well; discard the wash by shaking the contents of the wells into an approved biological waste container. Repeat the wash.
      6. Add 100 µl Calcein-AM Working Solution to each well.
      7. Incubate for 1 h at 37°C in a cell culture incubator.
      Measure the fluorescence in each well using a fluorescence plate reader at an excitation wavelength of ~485 nm and an emission wavelength ~520 nm.
      Assay characteristics and examples

      Figure 1: Relative cell attachment of various cell lines to laminin I, BMC, and BSA

      ~40,000 cells were added to wells containing laminin I, BMC, or BSA and incubated for 1.5 h at 37°C in a cell culture incubator in the presence of 6% CO2. Cells were washed gently with D-PBS and labeled with Calcein-AM for 1 h at 37°C in a cell culture incubator in the presence of 6% CO2. Fluorescence was measured as indicated in the Detailed Protocol. HT-1080 cells displayed appreciable binding to poly-L-lysine and served as a positive control (data not shown).


      Figure 2: Inhibition of HT-1080 cell attachment to laminin I

      ~10,000 cells were added to wells coated with laminin I in the presence or absence of inhibitory antibody, anti-integrin β1 (Cat. No. CP26). The cells were incubated and labeled and fluorescence was measured as indicated in Figure 1.
      Application referencesCaroll, D.K., et al. 2006. Nat. Cell Biol. 8, 551.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      InnoCyte™ and Interactive Pathways™ are trademarks of EMD Chemicals, Inc.