CytoSelect™ 48-well Cell Adhesion Assay (Fibrinogen, Colorimetric)

Details for Product No. ABIN2344821, Supplier: Log in to see
Reactivity
Human
Application
Cellular Assay (CA)
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'Independent Validation' Badge
Antigen Fibrinogen
Method validated Cellular Assay
Positive Control Ishikawa cells
Notes The Cell Adhesion Assay (Fibrinogen) ABIN2344821 successfully detects fibrinogen dependent cell adhesion.
Primary Antibody N/A
Secondary Antibody N/A
Protocol
  • Ishikawa cells cultured in DMEM-F12 medium and grown to 80% confluency.
  • Cells were trypsinised and re-suspended in serum-free medium.
  • A subset of cells was then incubated with RGD peptide for 30 min.
  • 0.5x106 cells of each cell population were added to fibrinogen coated wells and BSA control wells and incubated for 60 min.
  • The assay was performed according to kit’s manual.
  • Measurement:
  • FG, RGD 1FG, RGD 2FG, no-RGD 1FG, no-RGD 2BSA
    0.0790.0790.1020.1040.034
    0.0750.0730.0990.1040.038
    0.0720.0710.1100.1300.029
    0.0660.0690.1050.105
    Experimental Notes Fibrinogen coated wells resulted in higher absorbance readings than BSA well. Cells not exposed to RGD peptide displayed significantly higher absorbance values than cells previously exposed to RGD peptide: this confirms that integrins expressed on Ishikawa cells mediate adhesion to fibrinogen. Cells exposed to RGD peptide (and added to fibrinogen coated wells) displayed higher absorbance values than cells on BSA wells. This is consistent with the fact that integrins interact with proteins not exclusively in a RGD dependent fashion.
    Validation Images
    Cellular Assay validation image for CytoSelect™ 48-well Cell Adhesion Assay (Fibrinogen, Colorimetric) (ABIN2344821) Absorbance values at 560nm of 0.5x106 Ishikawa cells measured with the cel...
    Purpose The CytoSelect™ Cell Adhesion Assay Kit utilizes a Fibrinogen-coated 48-well plate (see Adhesion Plate Layout below). First, cells are seeded onto the coated substrate, where the adherent cells are captured. Next, unbound cells are washed away, and the adherent cells are fixed/stained. Finally, the stain is extracted and quantified colorimetrically.
    Brand CytoSelect™
    Sample Type Serum, Cell Samples
    Analytical Method Quantitative
    Detection Method Colorimetric
    Characteristics The CytoSelect™ Cell Adhesion Assay Kit provides a rapid, quantitative method for evaluating cell adhesion. The kit contains sufficient reagents for the evaluation of 48 samples (40 Human Fibrinogen-coated wells, 8 BSA-coated wells).
    Components
    1. Fibrinogen Adhesion Plate : One 48-well plate containing 40 Human Fibrinogen- coated wells and 8 BSA-coated wells (see layout below)
    2. Cell Stain Solution : One Bottle - 10.0 mL
    3. Extraction Solution : One Bottle - 10.0 mL 2
    Material not included
    1. Cell culture medium
    2. Serum free medium, such as DMEM containing 0.5 % BSA, 2 mM CaCl2 and 2 mM MgCl2
    3. Cell culture incubator (37 °C, 5 % CO2 atmosphere)
    4. 1X PBS containing 2 mM CaCl2 and 2 mM MgCl2
    5. Light microscope
    6. 96-well microtiter plate
    7. Microtiter plate reader
    Background Cell adhesion is a complex process involved in embryogenesis, migration/invasion, tissue remodeling, and wound healing. To perform these processes, cells adhere to extracellular matrix components (via adhesion receptors), forming complexes with components of the cytoskeleton that ultimately affect cell motility, differentiation, proliferation, and survival.
    Application Notes Optimal working dilution should be determined by the investigator.
    Comment

    • Full quantitation of cell adhesion with no manual cell counting
    • Plates precoated with uniform substrate layer of Fibrinogen

    Plate Pre-coated
    Assay Procedure
    1. Under sterile conditions, allow the Fibrinogen Adhesion Plate to warm up at room temperature for 10 minutes.
    2. Prepare a cell suspension containing 0.1-1.0 x 106 cells/mL in serum free media. Agents that inhibit or stimulate cell adhesion can be added directly to the cell suspension. 3
    3. Add 150 μL of the cell suspension to the inside of each well (BSA-coated wells are provided as a negative control).
    4. Incubate for 30-90 min in a cell culture incubator.
    5. Carefully discard or aspirate the media from each well (Note: Do not allow wells to dry). Gently wash each well 4-5 times with 250 μL PBS.
    6. Aspirate the PBS from each well and add 200 μL of Cell Stain Solution. Incubate for 10 minutes at room temperature.
    7. Discard or aspirate the Cell Stain Solution from the wells. Gently wash each well 4-5 times with 500 μL deionized water.
    8. Discard the final wash and let the wells air dry.
    9. Add 200 μL of Extraction Solution per well, and then incubate 10 minutes on an orbital shaker.
    10. Transfer 150 μL from each extracted sample to a 96-well microtiter plate and measure the OD 560nm in a plate reader.
    Restrictions For Research Use only
    Storage 4 °C
    Storage Comment Store all kit components at 4°C.
    Product cited in: Montealegre, La Rosa, Roh, Harvey, Murray: "The Enterococcus faecalis EbpA Pilus Protein: Attenuation of Expression, Biofilm Formation, and Adherence to Fibrinogen Start with the Rare Initiation Codon ATT." in: mBio, Vol. 6, Issue 3, pp. e00467-15, 2015 (PubMed).