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CBA083 InnoCyte™ 96-well Cell Adhesion Array

CBA083
  
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Fluorometric
      Description
      OverviewA cell adhesion array intended for the determination of the relative attachment of adherent cell lines to 6 different ECM proteins-laminin, fibronectin, vitronectin, and collagen type I, type III, and type IV.
      Catalogue NumberCBA083
      Brand Family Calbiochem®
      Application Data

      Approximately 30,000 cells were added to wells containing fibronectin, laminin I, collagen type IV, vitronectin, basement membrane complex, and 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 outlined in the Detailed Protocol.

      Approximately 30,000 cells were added to wells containing collagen I, III, or IV, 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 outlined in the Detailed Protocol.

      Approximately 10,000 cells were added to wells coated with laminin I in the presence or absence of inhibitory antibody, anti-integrin b1 (Cat. No. CP26). The cells were incubated and labeled and fluorescence was measured as outlined in Figure 1.
      Materials Required but Not Delivered• Cells and cell culture medium
      • Fluorescence reader capable of measuring fluorescence in 96-well plates at an excitation wavelength of ~485 nm and an emission wavelength of ~520 nm
      • 1X PBS for plate washing
      References
      ReferencesStupack, D.G. 2005. Cell Death and Differentiation 12, 1021.
      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.
      Frisch, S.M. 1994. J.Cell Biol. 124, 701.
      Meredith J.E. 1993. Mol. Biol. Cell 4, 953.
      Product Information
      Detection methodFluorometric
      Form96 Tests (14 samples x 6 ECM proteins and 12 negative controls)
      Format96-well plate
      Kit containsECM Protein-Coated 96-Well Plate Strips, Calcein-AM Solution, D-PBS, and a user protocol.
      Applications
      Biological Information
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 36/38

      Irritating to eyes and skin.
      S PhraseS: 26

      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Product Usage Statements
      Intended useThe Calbiochem® InnoCyte™ 96-Well Cell Adhesion Array is intended for the determination of the relative attachment of adherent cell lines to 6 different extracellular matrix (ECM) proteins: laminin, fibronectin, vitronectin, and collagens type I, type III, and type IV. BSA-coated wells serve as negative control.
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Irritant
      Storage Multiple storage requirements
      Storage ConditionsUpon arrival store the Calcein-AM at -20°C and the remaining components of the kit at 4°C.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsECM Protein-Coated 96-Well Plate Strips, Calcein-AM Solution, D-PBS, and a user protocol.
      Specifications

      Documentation

      InnoCyte™ 96-well Cell Adhesion Array Certificates of Analysis

      TitleLot Number
      CBA083

      References

      Reference overview
      Stupack, D.G. 2005. Cell Death and Differentiation 12, 1021.
      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.
      Frisch, S.M. 1994. J.Cell Biol. 124, 701.
      Meredith J.E. 1993. Mol. Biol. Cell 4, 953.
      User Protocol

      Revision17-December-2007 JSW
      Form96 Tests (14 samples x 6 ECM proteins and 12 negative controls)
      Format96-well plate
      Detection methodFluorometric
      StorageUpon arrival store the Calcein-AM at -20°C and the remaining components of the kit at 4°C.
      Intended useThe Calbiochem® InnoCyte™ 96-Well Cell Adhesion Array is intended for the determination of the relative attachment of adherent cell lines to 6 different extracellular matrix (ECM) proteins: laminin, fibronectin, vitronectin, and collagens type I, type III, and type IV. BSA-coated wells serve as negative control.
      BackgroundThe adhesion of cells to the ECM is a critical determinant of cytoskeletal organization and, thus, of cellular morphology. In addition to regulating cell shape, cell-matrix interactions also regulate cell proliferation, migration, and differentiation. Furthermore, cell-matrix interactions that support cytoskeletal organization of focal adhesions are essential for survival of anchorage-dependent, nontransformed cells. This wide range of activities suggests that the ECM is a key contributor to overall cellular physiology. Correspondingly, the fact that matricellular proteins modulate cell adhesion and cytoskeletal organization also suggests an important role for these proteins in essential processes. The most prominent members of adhesion receptor molecules on the cell surface are the integrins, which specifically interact with different ECM proteins. Adhesion receptors serve to mediate cell anchorage to the underlying ECM substrate and regulate cell morphology. Studies by Frisch and Francis termed the form of programmed cell death resulting from complete loss of substrate ECM contact 'anoikis'. Meredith et al., showed that substrate attachment without integrin engagement resulted in programmed cell death, while integrin-mediated attachment resulted in cell survival. Relevant ECM substrates and their corresponding integrin partners are are listed in the table below:

      Table 1: ECM Proteins and the Corresponding Integrin Receptors

      Principles of the assayThe InnoCyte™ Cell Adhesion Array is designed for the analysis of relative cell attachment to various ECM proteins. The kit is supplied with a 96-well strip plate coated with 6 different ECM proteins (two 8-well strips coated with laminin I, fibronectin, vitronectin, collagens type I, type III, and type IV, respectively) (see plate format below). Cells are seeded in the 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 for general attachment. Relative cell attachment is assessed using a fluorescence plate reader.
      Materials provided• ECM Protein-Coated 96-Well Plate Strips (Kit Component No. JA9413-1EA): 1 plate, 96 wells, supplied as 2 x 8-well strips coated with laminin I (LN), fibronectin (FN), vitronectin (VN), collagen I (CI), collagen III CIII), and collagen IV (CIV) in the following configuration:

      Table 2: ECM Proteins and the Corresponding Integrin Receptors


      • Calcein-AM Solution (Kit Component No. JA7705-50UL): 1 vial, 50 µl
      • D-PBS (JA7706-10ML): 1 vial, 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 of ~485 nm and an emission wavelength of ~520 nm
      • 1X PBS for plate washing
      Reagent preparation• Calcein-AM Working Solution: Prepare Calcein-AM Working Solution immediately prior to use. Note: the Calcein-AM Working Solution cannot be stored for future assays. Warm the Calcein-AM Solution and the D-PBS to room temperature. Dilute the 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 to the cell type (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 the required number of strips from the ECM protein-coated 96-Well Plate Strips 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 cell suspension (10,000-50,000/well) from step 2, in duplicate, to the designated wells of the ECM Protein-Coated 96-Well Plate Strips and incubate in a cell culture incubator for 1-2 h at 37°C.
      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 for a total of 2 washes.
      6. Add 100 µl Calcein-AM Working Solution to each well and incubate in a cell culture incubator for 1 hr at 37°C.
      7. Measure the fluorescence in each well using a fluorescence plate reader set at an excitation wavelength of ~485 nm and an emission wavelength ~520 nm.
      Assay characteristics and examples

      Figure 1: Relative Cell Attachment of Human Cell Lines

      Approximately 30,000 cells were added to wells containing fibronectin, laminin I, collagen type IV, vitronectin, basement membrane complex, and 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 outlined in the Detailed Protocol.



      Figure 2: Relative Attachment to Collagens

      Approximately 30,000 cells were added to wells containing collagen I, III, or IV, 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 outlined in the Detailed Protocol.


      Figure 3: Inhibition of HT-1080 Cell Attachment to Laminin I

      Approximately 10,000 cells were added to wells coated with laminin I in the presence or absence of inhibitory antibody, anti-integrin b1 (Cat. No. CP26). The cells were incubated and labeled and fluorescence was measured as outlined in Figure 1.

      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.