Chemotaxis assay procedure

Abstract

A chemotaxis assay procedure which is non-destructive of the cells being studied, which permits the ready performance of kinetic or time-dependent study of cell migration from the same sample, and which produces objective measurements includes the steps of:(a)labeling cells with a dye; (b) placing the labeled cells in a first chamber; (c) placing a chemical agent in a second chamber adjacent to said first chamber; (d) separating said first chamber from said second chamber with a radiation opaque membrane, said radiation opaque membrane having a plurality of substantially perpendicular transverse pores therein; (e) stimulating the labeled cells on the side of the membrane closest to said second chamber with electromagnetic radiation of a first wavelength whereby said labeled cells will emit electromagnetic radiation of a second wavelength; and (f) measuring the emitted electromagnetic radiation from the side of the radiation opaque membrane closest to the second chamber; wherein said radiation opaque membrane comprises a film which is not substantially transmissive to at least one of said first and second wavelengths of electromagnetic radiation. The radiation opaque membrane may comprise a dyed film or a film which has at least one radiation blocking layer applied thereto.

1. A non-destructive chemotaxis assay procedure comprising the steps of:
 a) labeling cells with a fluorescent dye;    b) placing the labeled cells in a first chamber;    c) placing a chemical agent in a second chamber adjacent to said first chamber, said chemical agent being capable of inducing migration of said labeled cells from said first chamber to said second chamber;    d) separating said first chamber from said second chamber with a radiation opaque membrane, said radiation opaque membrane having a plurality of substantially perpendicular transverse pores therein;    e) stimulating the labeled cells on the side of the membrane closest to said second chamber with electromagnetic radiation of a first wavelength whereby said labeled cells will emit electromagnetic radiation of a second wavelength; and    f) measuring the emitted electromagnetic radiation from the side of the radiation opaque membrane closest to the second chamber; wherein said radiation opaque membrane comprises a film which is not substantially transmissive to at least one of said first and second wavelengths of electromagnetic radiation.    
     
     
       2. The procedure of  claim 1 , wherein the fluorescent dye is Di-I. 
     
     
       3. The procedure of  claim 3 , wherein the radiation opaque membrane comprises a polyester film containing a blue dye. 
     
     
       4. The procedure of  claim 1 , wherein the radiation opaque membrane comprises a polycarbonate film coated with four layers of carbon and one layer of an admixture of gold and palladium. 
     
     
       5. The procedure of  claim 1 , wherein step (f) comprises measuring the electromagnetic radiation with a fluorescent plate reader. 
     
     
       6. The procedure of  claim 1 , further comprising the step of repeating steps (e) and (f) at least once at a predetermined time interval. 
     
     
       7. The procedure of  claim 6 , wherein the dye comprises a fluorescent dye. 
     
     
       8. The procedure of  claim 7 , wherein the fluorescent dye is Di-I. 
     
     
       9. The procedure of  claim 8 , wherein the radiation opaque membrane comprises a polyester film containing a blue dye. 
     
     
       10. The procedure of  claim 8 , wherein the radiation opaque membrane comprises a polycarbonate film coated with four layers of molecular carbon and one layer of an admixture of gold and palladium. 
     
     
       11. The procedure of  claim 7 , wherein the step (f) comprises measuring the electromagnetic radiation with a fluorescent plate reader. 
     
     
       12. The procedure of  claim 6 , wherein the film has a radiation blocking efficiency of at least approximately 95%. 
     
     
       13. The procedure of  claim 1 , wherein the film has a radiation blocking efficiency of at least approximately 95%. 
     
     
       14. The procedure of  claim 13 , wherein the film has a radiation blocking efficiency of at least approximately 97%. 
     
     
       15. A chemotaxis assay procedure comprising measuring the migration of cells across a radiation opaque membrane, wherein said procedure is non-destructive of said cells. 
     
     
       16. A cell migration assay procedure comprising measuring the migration of cells across a radiation opaque membrane wherein said procedure is non- destructive of said cells.   
     
     
       17. An assay procedure of  claim 16  including the further steps of:
   placing said cells in a first chamber;        labeling said cells in said first chamber;        separating said first chamber from said second chamber with said radiation opaque membrane; and        wherein said measuring step includes measuring cell presence in said second chamber by detecting said labeled cells in second chamber without substantially detecting said labeled cells in said first chamber.     
     
     
       18. An assay procedure of  claim 17  further including the step of inducing said migration of cells across said radiation opaque membrane. 
     
     
       19. An assay procedure of  claim 18  wherein said inducing step includes placing a chemical agent in said second chamber capable of creating a chemotactic reaction with said cells. 
     
     
       20. An assay procedure of  claim 17  wherein said labeling step includes labeling said cells with a die.