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 procedure of  claim 15  when said cells are selected from the group consisting of macrophages, eosinophils, fibroblasts, endothelial cells, epithelial cells, PMN's, tumor cells and prokaryotic organisms. 
     
     
       17. A procedure of  claim 15  including the step of inducing migration of said sample across said membrane. 
     
     
       18. A procedure of  claim 17  including the step of providing a chemotactic agent on one side of said membrane. 
     
     
       19. A procedure of  claim 18  including the steps of detecting sample presence on said one side of said membrane.