Methods of Detection of Changes in Cells

Abstract

Methods are provided to detect changes in cells without the use of detection labels.

We claim: 
     
         1 . A method of detection of responses of cells to stimuli comprising:
 (a) (i) immobilizing one or more extracellular matrix ligands to a surface of a colorimetric resonant reflectance biosensor or a grating-based waveguide biosensor, wherein the cells have cell surface receptors specific for the one or more extracellular matrix ligands; and adding the cells to the biosensor; or
 (ii) mixing cells with one or more extracellular matrix ligands, wherein the cells have cell surface receptors specific for the one or more extracellular matrix ligands; and adding the cells with one or more extracellular matrix ligands to a surface of a colorimetric resonant reflectance biosensor or a grating-based waveguide biosensor; 
   (b) exposing the cells to stimuli; and   (d) detecting the response of the cells to the stimuli.   
     
     
         2 . The method of  claim 1 , wherein the stimuli is a compound that modulates activity of a G protein-coupled receptor, an ion channel, P13 kinase, transient receptor potential channels, phospholipase C, receptor tyrosine kinases, cytokines, beta-arrestin pathway responses, cytoskeletal rearrangements, epigenetic signals, or signal transduction pathways. 
     
     
         3 . The method of  claim 1 , wherein the cells of (a) are in serum-free medium. 
     
     
         4 . The method of  claim 1 , wherein the detection of responses of cells to stimuli is augmented compared to methods where one or more extracellular matrix ligands are not included in the detection methodology. 
     
     
         5 . A method of preparing cells for an assay comprising:
 (a) washing cells with a buffered saline solution lacking calcium and magnesium;   (b) removing the buffered saline solution lacking calcium and magnesium from the cells;   (c) adding an isotonic ethylenediaminetetraacetate chealating agent to the cells;   (d) neutralizing the isotonic ethylenediaminetetraacetate chealating agent with buffered saline solution;   (e) washing the cells with buffered saline solution;   (f) adding the cells to a biosensor surface and allowing the cells to attach to the surface of the biosensor.   
     
     
         6 . The method of  claim 5 , wherein the cells are added to the biosensor surface in the presence of an adhesion modulator. 
     
     
         7 . The method of  claim 5 , wherein the biosensor surface has one or more extracellular matrix ligands immobilized to it or wherein the cells are mixed with one or more extracellular ligands before they are added to the biosensor surface. 
     
     
         8 . The method of  claim 7 , wherein binding of the cells to the extracellular matrix ligands is detected. 
     
     
         9 . A method of detection of responses of cells to stimuli comprising:
 (a) washing the cells with buffered saline solution;   (b) (i) immobilizing one or more extracellular matrix ligands to a surface of a colorimetric resonant reflectance biosensor or a grating-based waveguide biosensor, wherein the cells have cell surface receptors specific for the one or more extracellular matrix ligands; and adding the cells to the biosensor; or
 (ii) mixing cells with one or more extracellular matrix ligands, wherein the cells have cell surface receptors specific for the one or more extracellular matrix ligands; and adding the cells with one or more extracellular matrix ligands to a surface of a colorimetric resonant reflectance biosensor or a grating-based waveguide biosensor; 
   (c) exposing the cells to stimuli; and   (d) detecting the response of the cells to the stimuli.   
     
     
         10 . The method of  claim 9 , wherein prior to step (a), the following steps are performed:
 (i) washing cells with a buffered saline solution lacking calcium and magnesium;   (ii) removing the buffered saline solution lacking calcium and magnesium from the cells;   (iii) adding an isotonic ethylenediaminetetraacetate chealating agent to the cells;   (iv) neutralizing the isotonic ethylenediaminetetraacetate chealating agent with buffered saline solution.   
     
     
         11 . The method of  claim 9 , wherein the cells are added to the biosensor surface in the presence of an adhesion modulator. 
     
     
         12 . The method of  claim 9 , wherein the stimuli is a compound that modulates activity of a G protein-coupled receptor, an ion channel, P13 kinase, transient receptor potential channels, phospholipase C, receptor tyrosine kinases, cytokines, beta-arrestin pathway responses, cytoskeletal rearrangements, epigenetic signals, or signal transduction pathways. 
     
     
         13 . A method of determining if a test reagent affects movement of cells comprising:
 (a) immobilizing one or more extracellular matrix protein ligands to the bottom of a well of a microtiter plate, wherein the bottom of the well comprises a colorimetric resonant reflectance biosensor or a grating-based waveguide biosensor, and wherein the well has a microfluidic channel that can deliver a test reagent to one location within the well;   (b) adding cells that have cell surface receptors specific for the one or more extracellular matrix protein ligands to the well;   (c) delivering a test reagent to the well using the microfluidic channel; wherein the test reagent is a suspected chemotactic agent;   (d) detecting movement of the cells in the well, wherein if movement of the cells in the well is detected, then the test reagent affects movement of the cells.   
     
     
         14 . The method of  claim 13 , wherein the test reagent is a compound that modulates activity of a G protein-coupled receptor, an ion channel, P13 kinase, transient receptor potential channels, phospholipase C, receptor tyrosine kinases, cytokines, beta-arrestin pathway responses, cytoskeletal rearrangements, epigenetic signals, or signal transduction pathways. 
     
     
         15 . A method of determining if a test compound affects specific binding of cells to extracellular matrix ligands comprising:
 (a) adding first cells to a first colorimetric resonant reflectance biosensor surface or a first grating-based waveguide biosensor surface, wherein the biosensor surface comprises immobilized extracellular matrix ligands, wherein the first cells have cell surface receptors specific for the extracellular matrix ligands, allowing the first cells to attach to the surface of the first biosensor, and determining a peak wavelength value or signal for the first cells;   (b) adding second cells to a second colorimetric resonant reflectance biosensor surface or a second grating-based waveguide biosensor surface, wherein the second biosensor surface comprises immobilized extracellular matrix ligands, wherein the second cells have cell surface receptors specific for the extracellular matrix ligands, adding a test compound to the biosensor surface, allowing the second cells to attach to the surface of the biosensor, and determining a peak wavelength value or signal for the second cells; and   (c) comparing the peak wavelength value or signal obtained for the first cells to the peak wavelength value or signal for the second cells; wherein if the peak wavelength value or signal for the second cells is substantially different from the peak wavelength value or signal for the first cells, then the test compound affects specific binding of cells to extracellular matrix ligands.   
     
     
         16 . The method of  claim 15 , wherein the test compound is a compound that modulates activity of a G protein-coupled receptor, an ion channel, P13 kinase, transient receptor potential channels, phospholipase C, receptor tyrosine kinases, cytokines, beta-arrestin pathway responses, cytoskeletal rearrangements, epigenetic signals, or signal transduction pathways. 
     
     
         17 . A method of analyzing cell changes in real time at comprising:
 (a) immobilizing one or more extracellular matrix ligands to the surface of a colorimetric resonant reflectance biosensor or a grating-based waveguide biosensor;   (b) adding cells to the biosensor, wherein the cells have cell surface receptors specific for the one or more extracellular matrix ligands;   (c) exposing the cells to stimuli; and   (d) detecting the response of the cells to the stimuli using a detection device with a resolution of about 2 to 10 micrometers.   
     
     
         18 . The method of  claim 17 , wherein the stimuli is a compound that modulates activity of a G protein-coupled receptor, an ion channel, P13 kinase, transient receptor potential channels, phospholipase C, receptor tyrosine kinases, cytokines, beta-arrestin pathway responses, cytoskeletal rearrangements, epigenetic signals, or signal transduction pathways. 
     
     
         19 . The method of  claim 17 , wherein the cell changes are changes in changes in cell growth patterns, cell death patterns, cell movement, cell size or volume, or cell adhesion. 
     
     
         20 . A method of detecting changes in cell responses comprising:
 (a) immobilizing one or more extracellular matrix ligands to the surface of a colorimetric resonant reflectance biosensor or a grating-based waveguide biosensor;   (b) adding cells to the biosensor in serum-free medium, wherein the cells have cell surface receptors specific for the one or more extracellular matrix ligands;   (c) exposing the cells to stimuli without washing off the serum free-medium; and   (d) detecting the response of the cells to the stimuli.   
     
     
         21 . The method of  claim 20 , wherein the stimuli is a compound that modulates activity of a G protein-coupled receptor, an ion channel, P13 kinase, phospholipase C, receptor tyrosine kinases, cytokines, beta-arrestin pathway responses, cytoskeletal rearrangements, epigenetic signals, or signal transduction pathways. 
     
     
         22 . A colorimetric resonant reflectance biosensor or a grating-based waveguide biosensor surface, wherein the biosensor is prepared by applying two or more types of extracellular matrix ligands, and ovalbumin or fetal bovine serum to a surface of the biosensor and drying the surface of the biosensor. 
     
     
         23 . A method of identifying a compound that affects adhesion of cells comprising:
 (a) (i) immobilizing one or more extracellular matrix ligands to a surface of a colorimetric resonant reflectance biosensor or a grating-based waveguide biosensor, wherein the cells have cell surface receptors specific for the one or more extracellular matrix ligands; and adding the cells to the biosensor; or
 (ii) mixing cells with one or more extracellular matrix ligands, wherein the cells have cell surface receptors specific for the one or more extracellular matrix ligands; and adding the cells with one or more extracellular matrix ligands to a surface of a colorimetric resonant reflectance biosensor or a grating-based waveguide biosensor; 
   (b) treating the cells with a test compound;   (c) detecting a colorimetric resonant reflectance optical first PWV or signal and comparing the first PWV or signal to a second PWV or signal from control cells that were not treated with the test compound to determine if the test compound affects adhesion of the cells.   
     
     
         24 . The method of  claim 23 , wherein the test compound is a compound that modulates activity of a G protein-coupled receptor, an ion channel, P13 kinase, transient receptor potential channels, or phospholipase C. 
     
     
         25 . The method of  claim 23 , wherein after (b) an adhesion modulator is added to the cells, wherein the control cells are also treated with the adhesion modulator. 
     
     
         26 . A method of identifying a modified extracellular matrix (ECM) ligand that affects adhesion of cells comprising:
 (a) applying cells to a surface of a colorimetric resonant reflectance optical biosensor or a grating-based waveguide biosensor, wherein modified ECM ligands are immobilized to the surface of the biosensor;   (b) adding an adhesion modulator to the cells;   (c) detecting a colorimetric resonant reflectance optical first PWV or signal for the cells and comparing the first PWV or signal to a second PWV or signal from control cells that were added to a surface of a colorimetric resonant reflectance optical biosensor or the grating-based waveguide biosensor, wherein non-modified ECM ligands are immobilized to the surface of the biosensor to determine if the test compound affects adhesion of the cells.   
     
     
         27 . A method of determining if a transfected cell produces a recombinant protein comprising:
 (a) immobilizing one or more extracellular matrix ligands to the surface of a colorimetric resonant reflectance biosensor or a grating-based waveguide biosensor;   (b) adding cells that have been transfected with a nucleic acid molecule that encodes a recombinant protein and that have cell surface receptors specific for the one or more extracellular matrix ligands to the surface of the biosensor;   (c) determining a first PWV or signal for the cells;   (e) adding a modulator that causes a differential response in the transfected cells that expresses the recombinant protein as compared to transfected cells that do not express the recombinant protein;   (e) determining a second PWV or signal for the cells;   (f) comparing the first peak wavelength value or signal to the second peak wavelength value or signal;   wherein if the second peak wavelength value or signal is substantially different from the first peak wavelength value or signal, then the transfected cells express the recombinant protein.