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.

Description:
CROSS- REFERENCE TO RELATED APPLICATIONS    
     This application is a continuation of U.S. Ser. No.  09 / 472 , 490 , filed Dec.  23 ,  1999 , which is a reissue application Ser. No.  09 / 159 , 427  filed Sep.  23 ,  1998 , now abandoned, which is a reissue of application Ser. No.  08 / 383 , 058  filed Feb.  3 ,  1995  U.S. Pat. No.  5 , 601 , 997 . 
    
    
     FIELD OF THE INVENTION 
     This invention relates to a chemotaxis assay procedure and, more particularly, relates to an in vitro chemotaxis assay procedure which is non-destructive of the cell sample and permits kinetic study of the chemotactic response. This invention also relates to a novel radiation opaque membrane for use in the chemotaxis procedure. 
     BACKGROUND OF THE INVENTION 
     Chemotaxis is broadly defined as the orientation or movement of an organism or cell in relation to a chemical agent. Chemotaxis assays, particularly in vitro chemotaxis assays, are widely used procedures in medical, biological, pharmaceutical and toxicological research. Such assays are perhaps most widely used to determine the effect of a chemical agent on the inflammatory process, either as a stimulant or inhibitor of that process. 
     The currently used chemotaxis assay procedure derives from that originally developed by S. Boyden in 1962. (See, S. Boyden, The Chemotactic Effect of Mixtures of Antibody and Antigen on Polymorphonuclear Leucocytes, J. Exp. Med. 115: pp. 453-466, 1962). Essentially, the procedure involves placing a suspension of neutrophils and a chemical agent in two separate chambers, which chambers are separated by a polycarbonate filter. The neutrophils are typically either human polymorphonuclear neutrophils (“PMN&#39;s”) prepared from the peripheral blood of volunteers or PMN&#39;s prepared from the peritoneal fluid of animals, such as guinea pigs or rabbits. 
     After a predetermined period of time, the filter is removed and cells on the filter surface closest to the chamber containing the cell suspension are carefully removed. The remaining cells on the filter are then fixed and stained. Using a high power microscope, the filter is examined and the number of cells appearing on the underside of the filter (i.e., the side of the filter closest to the chamber containing the chemical agent) are counted manually. A positive chemotactic response is indicated by the cells having migrated or “crawled” through the filter to the side closest to the chamber containing the chemical agent. Because of the time required to do so, typically the entire filter is not examined. Rather, representative sample areas are examined and counted. 
     There are several disadvantages to this procedure. The examination and counting of the cells on the filter is time-consuming, tedious and expensive. It is also highly subjective because it necessarily involves the exercise of judgment in determining whether to count a cell that has only partially migrated across the filter. In addition, the time and expense associated with examining the entire filter necessitates that only representative areas, selected at random, be counted, thus rendering the results less accurate than would otherwise be the case if the entire filter were examined and counted. 
     Perhaps the most important disadvantage in this procedure is that the fixing step kills the cells. That is, the procedure is destructive of the cell sample. Thus, in order to determine a time-dependent relationship of the chemotactic response; that is, a kinetic study, of a particular chemical agent, it is necessary to run multiple samples for each of multiple time periods. When one considers that multiple samples, as well as positive and negative controls, are necessary to obtain reliable data, a single chemotaxis assay can produce dozens of filters, each of which needs to be individually examined and counted. The time and expense associated with a time-dependent study is usually prohibitive of conducting such a study using the Boyden procedure. 
     Alternatives to the Boyden assay have been proposed to overcome some of the above disadvantages. See generally, P. Wilkinson, Micropore Filter Methods for Leukocyte Chemotaxis, Methods in Enzymology, Vol. 162, (Academic Press, Inc. 1988), pp. 38-50. See also, Goodwin, U.S. Pat. No. 5,302,515; Guiruis et al., U.S. Pat. No. 4,912,057; Goodwin, U.S. Pat. No. 5,284,753; and Goodwin, U.S. Pat. No. 5,210,021. Although the chemotaxis devices and procedures described in these references have some advantages over the original Boyden procedure and apparatus, they are not without their shortcomings. For example, all of these procedures, like Boyden, require that the filter be removed and the non-migrated cells wiped or brushed from the filter before the migrated cells can be counted. In addition, most of these procedures require fixing and staining the cells and none of them permit the kinetic or time-dependent study of the chemotactic response of the same cell sample. 
     SUMMARY OF THE INVENTION 
     I have developed a chemotaxis assay procedure which avoids the above disadvantages, which is non-destructive, and which readily permits kinetic study of the chemotactic response. The chemotaxis procedure of this invention is simple, quick and inexpensive to perform, produces objective data, and is usable with a variety of different cell types. 
     Basically, the non-destructive chemotaxis assay procedure comprises 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.       

     In another aspect, the invention comprises a radiation opaque membrane for used in a chemotaxis assay procedure wherein cells labeled with a dye are stimulated with electromagnetic radiation of a first wavelength whereby the cells will emit electromagnetic radiation of a second wavelength, said radiation opaque membrane comprising a film which is not substantially transmissive to at least one of said first or second wavelengths of electromagnetic radiation, said radiation opaque membrane having a plurality of substantially perpendicular transverse pores therein. 
     These and other aspects of the invention will become apparent upon a reading of the following detailed description of the embodiments, with reference to the drawings, and the appended claims. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a perspective view of a preferred apparatus used in carrying out the present procedure. 
         FIG. 2  is an enlarged, sectional view of the apparatus of  FIG. 1  as seen along line  2 — 2  of FIG.  1 . 
         FIG. 3A  is a simplified schematic view, in cross-section, of cells migrating across one embodiment of the radiation opaque membrane of the present invention. 
         FIG. 3B  is a simplified schematic view, in cross-section, of cells migrating across another embodiment of the radiation opaque membrane of the present invention. 
         FIGS. 4-7  are graphs of fluorescence units vs. incubation time of the chemotaxis data generated by the Examples. 
     
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
     Although not critical to the present invention, a description of the preferred apparatus for use in carrying out the chemotaxis procedure of this invention is included because it is believed to be helpful in illustrating the advantages of this invention over the prior art. It is to be expressly understood, however, that any number of devices may be used in carrying out the present procedure and the invention is not limited to the use of any particular apparatus, except as set forth in the appended claims. 
     With reference first being made to  FIGS. 1 and 2 , the preferred apparatus comprises a multi-well culture plate which is widely available from a variety of commercial sources. This type of apparatus is commonly employed to study the effects of chemical agents on cell growth. As seen in  FIGS. 1 and 2 , the apparatus comprises a plate  20  having a plurality of spaced-apart wells  22 . Each well  22  is provided with an insert  24  adapted to fit inside the well. In the parlance of this specification, the interior of the insert comprises one chamber and the exterior of the insert comprises a second chamber. The size, shape and number of wells  22 , inserts  24 , and plate  20  are not critical to this invention. 
     For purposes of this invention, the bottom of the insert  24  has been provided with a radiation opaque membrane  10  of this invention, which separates the two chambers. The radiation opaque membrane  10  may be attached to the bottom of the insert by any conventional means, such as glue or other adhesive, heat welding, ultrasonic welding, etc. In practice, the labeled cells are placed in the insert  24  and the chemical agent is placed in the well  22 . The chemotactic reaction will cause the labeled cells to migrate or “crawl” from the chamber  24  to chamber  22 , through the pores  16  in the radiation opaque membrane  10 , as particularly shown in  FIGS. 3A and 3B . 
     As seen in  FIG. 2 , a space  28  is created between the radiation opaque membrane  10  and the bottom of the well  22 . A distance of about 1 mm between the bottom of well  22  and the radiation opaque membrane  10  is generally sufficient to permit the free migration of cells across the radiation opaque membrane. The space  28  may be conveniently created by providing the insert  24  with stand-offs  26 , which may take any convenient form or shape (e.g. legs, bosses, flange, etc.). When using stand-offs, care should be taken not to isolate the fluid in space  28  from the remainder of the fluid in the well  22 , which would tend to create a separate concentration gradient in the space  28 . Alternatively, the space  28  may be created by suspending the insert  24  within the well  22  by the use of, for example, radial projections  27  which rest on the surface of plate  20  as shown in  FIGS. 1 and 2 . 
     At predetermined periods, the quantum of cells that have migrated across the radiation opaque membrane will be determined by first exciting or stimulating the labeled cells on the side of the radiation opaque membrane  10  closest to the chamber  22  and measuring the radiation emitted by those labeled cells. With the preferred apparatus illustrated in  FIGS. 1 and 2 , this step would comprise stimulating and measuring the radiation from below the radiation opaque membrane  10 , that is, through space  28 . It will be understood by those skilled in the art that it is preferred that at least the chamber through which the stimulation and measurement of radiation will take place is substantially transparent to both the radiation being measured and any radiation needed to excite or stimulate the dye used to label the cells. In the preferred embodiment, the apparatus is made of a clear, transparent material, such as polystyrene, polycarbonate, LUCITE®, glass, etc. 
     The device  30  used to stimulate the cells and measure the emitted radiation will, of course, depend on the dye used to label the cells and the type of apparatus used for the assay procedure. For example, if the plate apparatus of  FIGS. 1 and 2  is used, a fluorescent plate reader, such as a Cytofiuor™ 2300 (Millipore Corp., Marlborough, MA), can be used to advantage. The radiation opaque membrane  10  will substantially prevent either the stimulation of the cells in chamber  24  or the transmission of radiation from the cell sample in chamber  24  into the space  28 , or will prevent both. Accordingly, the radiation measured will provide a direct quantitative measure of the number of cells that have migrated across the radiation opaque membrane  10  from chamber  24  to chamber  22 . 
     It will be appreciated by those skilled in the art that neither insert  24 , nor radiation opaque membrane  10 , nor the non-migrated cells adhered to it, need be removed prior to measuring the radiation corresponding to the migrated cells. This permits repeated measurements of the chemotactic response of the same cell sample, thus permitting simple and rapid quantitative determinations in a kinetic, or time-dependent, profile of the chemotactic response with a minimum number of test samples. In addition, the devices used to measure the radiation, such as plate readers or spectrophotometers, are highly sensitive and accurate pieces of equipment and provide objective data corresponding to the number of migrated cells. This is a distinct advantage over the prior art procedures which rely upon subjective physical examination under a microscope. 
     As mentioned above, the chemotaxis assay of this invention can be used with a variety of cell types. Examples include, but are not limited to, macrophages, eosinophils, fibroblasts, endothelial cells, epithelial cells, PMN&#39;s, tumor cells and prokaryotic organisms. The only practical limitations on the cell type are its ability to exhibit a chemotactic response and its ability to be labeled. 
     In accordance with the present invention, the cell sample is labeled with a fluorescent dye. The process of labeling cells with dyes is well known, as is the variety of fluorescent dyes that may be used for labeling particular cell types. See e.g. R. Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc. (1989). A particularly preferred fluorescent dye for use with an HL-60 cell line (ATCC No. CCL 240) in the present invention is Di-I (Molecular Probes, Inc.; Eugene, OR). 
     It should be mentioned here that, in theory, non-fluorescent dyes may be used in the present invention. At the present time however, there are no known devices that can be used to measure the transmitted light from migrated cells to the exclusion of the transmitted light from the non-migrated cells. Accordingly, the practical utility of using non-fluorescent dyes in the present invention awaits the discovery or invention of such a device. 
     A particularly novel aspect of the present invention is the use of a radiation opaque membrane which is not substantially transmissive to at least the wavelength of electromagnetic radiation used to stimulate the labeled cells or the wavelength of electromagnetic radiation emitted by the labeled cells. Preferably, the radiation opaque membrane is not substantially transmissive to both wavelengths of electromagnetic radiation, which would protect against excitation of non-migrated cells and would also prevent transmission of radiation emitted by any non-migrated cells that may, nevertheless, become stimulated. It may be advantageous in certain situations, such as for example where mixed cell types and multiple labeling dyes are used, to selectively block either the excitation wavelength or the emission Wavelength. Because the radiation opaque membrane is porous, it will be impossible to completely block all transmission of radiation across the radiation opaque membrane, simply because some radiation will be transmitted through the pores in the radiation opaque membrane. In practice, however, the quantum of radiation so transmitted will be relatively constant and negligible in terms of the quantum of radiation radiating from the migrated cells. Generally speaking, however, the radiation opaque membrane (absent any pores) should have a blocking efficiency of at least approximately 95%. That is, the membrane should be capable of blocking at least approximately 95% of the intended radiation, either the radiation used to stimulate the cells, the radiation emitted by the labeled cells, or the combined stimulation and emission radiation. 
     In accordance with the present invention, such membranes permit the measurement of radiation emitted from the labeled cells that have migrated through the radiation opaque membrane without interference from radiation emitted from the labeled cells that have not migrated, without the need to remove the non-migrated cells from the radiation opaque membrane. This is a significant advantage of the present invention over the prior art procedures, not only because it avoids the tedious steps of removing the filter and scraping the non-migrated cells from the filter, but also because it is non-destructive of the cell sample and thus permits repeated measurements of the same test sample at different time intervals. 
     The radiation opaque membrane itself may be of any convenient construction, so long as it has the properties mentioned above. In general, the radiation opaque membrane  10  comprises a non-fibrous film  12  of polyester, polycarbonate, polyethylene terephthalate, polylactic acid, nylon, etc. Depending on the type of film used, the film may be dyed to obtain the radiation blocking properties discussed above. In lieu of or in addition to using a dyed film, one or more radiation blocking layers  14  may be applied to the film by any conventional process suitable for the particular film and blocking layer(s) being used, such as coating under vacuum, layer transfer, sputtering, spin coating, vacuum deposition, etc. The thickness of the radiation opaque membrane  10  is not critical to the invention. Membranes having a thickness in the range customarily used in the art are suitable for use herein. 
     As already noted, the radiation opaque membrane must have a plurality of pores  16  disposed substantially perpendicular to the plane of the radiation opaque membrane to permit the migration of cells across the radiation opaque membrane. The diameter of the pores is not particularly critical and, to a large extent, depends upon the size of the cells being studied. Generally speaking, the pores  16  must be of such diameter to prevent the cells from passively traversing the radiation opaque membrane while at the same permitting the cells to actively “crawl” through the radiation opaque membrane. It is readily within the skill of the ordinary artisan to determine the appropriate pore size for a particular chemotaxis assay without undue experimentation. Pores of suitable size can be provided in the film by any known process, such as atomic etching. If a radiation blocking layer(s) is to be applied to the film, it may be done either before or after the pores have been provided. 
     EXAMPLES 
     Cell Sample 
     The cell line HL-60 (ATCC No. CCL 240) was maintained in logarithmic growth phase as a suspension culture at about 10 6  cells/mL. in RPMI 1640 medium (Mediatech Cellgrow, Fisher Scientific, Pittsburgh, PA.) supplemented with 20% (volume by volume) fetal bovine serum. (Hyclone Laboratories, Salt Lake City, UT). The cells were differentiated into mature myelocytes and neutrophils by incubating the cells for 48 hours at 37° C. in media containing 1.5% (volume by volume) dimethylsulfoxide. 
     Cell Labeling 
     Following the treatment with dimethylsulfoxide, the cells were incubated with 50 μM Di-I fluorescent dye (Molecular Probes, Inc., Eugene, OR) at room temperature for 0.5-2 hours. The cells were then washed with Hanks&#39; Balanced Salt Solution (“HBSS”) (Sigma Chemical Co., St. Louis, MO.) and re-suspended in HBSS to achieve a cell concentration of 10 6  cells/mL. The fluorescence of 0.5 mL. of cell suspension was measured in a Cytofluor™ 2300 fluorescent plate reader (Millipore Corp., Marlborough, MA.). 
     Membrane Preparation 
     Membrane 1: Polycarbonate film having a plurality of pores of 8 μm diameter were coated with four molecular layers of carbon and one layer of an admixture of gold and palladium in a vacuum evaporator. The resulting radiation opaque membrane had a thickness of about 10 μm and was approximately 97% efficient in blocking the combined stimulation and emission radiation. 6 mm disks of the radiation opaque membrane were glued to the bottom of inserts similar to the Millicell HA-12 mm (Millipore Corp.) or the Transwell-6.5 mm (Costar Corp., Cambridge, MA.) inserts with clear silicone rubber cement. 
     Membrane 2: A non-porous polyester film (18 μm thick) containing a blue dye (Aquired Technology Inc., Alpharetta, GA.) was subjected to atomic etching to produce a 10 μm thick radiation opaque membrane containing a plurality of pores of 8 μm diameter having a combined radiation blocking efficiency of approximately 99%. 6 mm disks of the radiation opaque membrane were glued to the bottoms of inserts as with membrane 1. 
     Test Procedure 
     Each insert equipped with the either membrane 1 or membrane 2 were placed in a well of a 24-well culture plate (Falcon, Fisher Scientific). 0.5 mL of labeled cell suspension was placed inside each insert. The plate was incubated for 30 minutes at 37° C. to allow the cells to settle on the radiation opaque membrane. The fluorescence of each well was then measured with the Cytofluor™ 2300 to obtain a zero time reading. 0.5 mL of either N-formyl methionyl leucyl phenylalanine (“f-MLP”) (Sigma Chemical Co.) or HBBS was then added to each well. The fluorescence in each well was then measured at periodic time intervals using the Cytofluor™ 2300 at sensitivity setting 4. Results using membrane 1 are reported in Tables 1 and 2 and graphically illustrated in  FIGS. 4 and 5 . Results using membrane 2 are reported in Table 3 and graphically illustrated in  FIGS. 6 and 7 . 
     
       
         
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Well 
                   
                   
               
               
                 Num- 
                 Test Solutions 
                 Fluorescence 
               
             
          
           
               
                 ber 
                 well/insert 
                 0 hr. 
                 1 hr. 
                 2 hr. 
                 3 hr. 
                 4 hr. 
                 5 hr. 
               
               
                   
               
             
          
           
               
                 1 
                 HBSS/HBSS 
                 546 
                 757 
                 862 
                 922 
                 927 
                 904 
               
               
                 2 
                 HBSS/f-MLP 1   
                 383 
                 1046 
                 1355 
                 1433 
                 1370 
                 1359 
               
               
                 3 
                 f-MLP 2 /f-MLP 
                 706 
                 654 
                 708 
                 732 
                 728 
                 753 
               
               
                 4 
                 f-MLP 3 /f-MLP 
                 467 
                 412 
                 435 
                 460 
                 447 
                 454 
               
               
                 5 
                 Blank 
                 130 
                 124 
                 125 
                 125 
                 125 
                 125 
               
               
                 6 
                 Blank 
                 132 
                 127 
                 127 
                 128 
                 127 
                 126 
               
               
                 7 
                 Blank 
                 131 
                 127 
                 127 
                 128 
                 127 
                 126 
               
               
                 8 
                 Blank 
                 128 
                 124 
                 126 
                 125 
                 127 
                 125 
               
               
                 9 
                 Blank 
                 129 
                 125 
                 126 
                 126 
                 126 
                 125 
               
               
                 10 
                 Blank 
                 130 
                 127 
                 127 
                 127 
                 128 
                 127 
               
               
                 11 
                 Blank 
                 135 
                 133 
                 132 
                 132 
                 132 
                 132 
               
               
                 12 
                 Blank 
                 130 
                 126 
                 125 
                 126 
                 125 
                 125 
               
               
                 13 
                 Blank 
                 132 
                 128 
                 129 
                 130 
                 129 
                 128 
               
               
                 14 
                 Blank 
                 134 
                 141 
                 136 
                 139 
                 136 
                 137 
               
               
                 15 
                 Blank 
                 137 
                 134 
                 133 
                 134 
                 134 
                 132 
               
               
                 16 
                 Blank 
                 136 
                 131 
                 132 
                 133 
                 132 
                 132 
               
               
                 17 
                 Blank 
                 135 
                 134 
                 132 
                 134 
                 131 
                 132 
               
               
                 18 
                 Blank 
                 137 
                 132 
                 131 
                 132 
                 132 
                 133 
               
               
                 19 
                 Blank 
                 136 
                 132 
                 131 
                 132 
                 132 
                 133 
               
               
                 20 
                 Blank 
                 139 
                 135 
                 132 
                 135 
                 134 
                 135 
               
               
                 21 
                 Blank 
                 141 
                 135 
                 136 
                 138 
                 136 
                 137 
               
               
                 22 
                 Blank 
                 140 
                 137 
                 137 
                 138 
                 136 
                 137 
               
               
                 23 
                 0.5 mL cells 
                 9999 3   
                 9999 
                 9999 
                 9999 
                 9999 
                 9999 
               
               
                 24 
                 0.5 mL cells 
                 9999  
                 9999 
                 9999 
                 9999 
                 9999 
                 9999 
               
               
                   
               
               
                 Notes:  
               
               
                   1 Conc. = 10 −7  M  
               
               
                   2 f-MLP added to cell suspension immediately before start of experiment.  
               
               
                   3 Fluorescence was greater than measurable at selected sensitivity setting.  
               
             
          
         
       
     
     
       
         
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Well 
                   
                 FLUORESCENCE 
               
             
          
           
               
                 Num- 
                 Test Solutions 
                 0 
                 15 
                 30 
                 60 
                 90 
                 120 
                 150 
               
               
                 ber 
                 insert/well 
                 min. 
                 min. 
                 min. 
                 min. 
                 min. 
                 min. 
                 min. 
               
               
                   
               
             
          
           
               
                 1 
                 HBSS/f-MLP 1   
                 2927 
                 4195 
                 4475 
                 4642 
                 4761 
                 4801 
                 4788 
               
               
                 2 
                 HBSS/f-MLP 
                 2895 
                 4165 
                 4400 
                 4539 
                 4642 
                 4681 
                 4655 
               
               
                 3 
                 HBSS/f-MLP 
                 2631 
                 3398 
                 3584 
                 3645 
                 3728 
                 3759 
                 3728 
               
               
                 4 
                 HBSS/f-MLP 
                 2594 
                 3446 
                 3707 
                 3813 
                 3932 
                 3988 
                 3999 
               
               
                 5 
                 HBSS/f-MLP 
                 2515 
                 3388 
                 3594 
                 3614 
                 3717 
                 3759 
                 3770 
               
               
                 6 
                 f-MLP 2 /F-MLP 
                 2854 
                 2675 
                 2721 
                 2721 
                 2783 
                 2783 
                 2783 
               
               
                 7 
                 HBSS/HBSS 
                 2558 
                 2683 
                 2736 
                 2783 
                 2862 
                 2886 
                 2911 
               
               
                 8 
                 HBSS/HBSS 
                 2862 
                 2977 
                 3028 
                 3053 
                 3114 
                 3132 
                 3105 
               
               
                 9 
                 HBSS/HBSS 
                 3105 
                 3194 
                 3221 
                 3220 
                 3294 
                 3313 
                 3294 
               
               
                 10 
                 HBSS/HBSS 
                 2377 
                 2660 
                 2767 
                 2846 
                 2927 
                 2952 
                 2960 
               
               
                 11 
                 Blank 
                 165 
                 163 
                 162 
                 163 
                 160 
                 160 
                 160 
               
               
                 12 
                 Blank 
                 166 
                 163 
                 164 
                 162 
                 161 
                 160 
                 657 
               
               
                 13 
                 Blank 
                 166 
                 163 
                 163 
                 163 
                 158 
                 161 
                 157 
               
               
                 14 
                 Blank 
                 166 
                 166 
                 163 
                 163 
                 163 
                 162 
                 159 
               
               
                 15 
                 Blank 
                 162 
                 160 
                 160 
                 159 
                 156 
                 157 
                 156 
               
               
                 16 
                 Blank 
                 163 
                 160 
                 159 
                 159 
                 156 
                 157 
                 153 
               
               
                 17 
                 Blank 
                 162 
                 161 
                 160 
                 159 
                 158 
                 156 
                 156 
               
               
                 18 
                 Blank 
                 164 
                 161 
                 162 
                 159 
                 158 
                 151 
                 147 
               
               
                 19 
                 Blank 
                 163 
                 161 
                 162 
                 158 
                 158 
                 158 
                 154 
               
               
                 20 
                 Blank 
                 162 
                 161 
                 160 
                 159 
                 158 
                 153 
                 145 
               
               
                 21 
                 Blank 
                 168 
                 166 
                 165 
                 163 
                 163 
                 163 
                 160 
               
               
                 22 
                 Blank 
                 165 
                 164 
                 163 
                 159 
                 160 
                 151 
                 151 
               
               
                 23 
                 Blank 
                 171 
                 168 
                 168 
                 164 
                 150 
                 166 
                 162 
               
               
                 24 
                 Blank 
                 172 
                 170 
                 169 
                 153 
                 151 
                 162 
                 162 
               
               
                   
               
               
                 Notes:  
               
               
                   1 Conc. = 10 −8  M  
               
               
                   2 f-MLP added to cell suspension immediately before start of experiment  
               
             
          
         
       
     
     
       
         
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Well 
                 Test Solutions 
                 Fluorescence 
               
             
          
           
               
                 Number 
                 insert/well 
                 0 hr. 
                 0.5 hr. 
                 1 hr. 
                 2 hr. 
                 3 hr. 
               
               
                   
               
             
          
           
               
                 1 
                 HBSS/HBSS 1   
                 1079 
                 1378 
                 1586 
                 1770 
                 1810 
               
               
                 2 
                 HBSS/HBSS 
                  891 
                 1058 
                 1194 
                 1351 
                 1421 
               
               
                 3 
                 HBSS/HBSS 
                  940 
                 1221 
                 1382 
                 1533 
                 1617 
               
               
                 4 
                 Blank 
                  178 
                 169 
                 169 
                 167 
                 166 
               
               
                 5 
                 f-MLP 2 /f-MLP 
                  961 
                 1245 
                 1390 
                 1564 
                 1711 
               
               
                 6 
                 0.3 mL cells 
                 9999 
                 9999 
                 9999 
                 9999 
                 9999 
               
               
                 7 
                 HBSS/f-MLP 3   
                 1055 
                 1770 
                 2066 
                 2351 
                 2536 
               
               
                 8 
                 HBSS/f-MLP 
                 1064 
                 1454 
                 1846 
                 2143 
                 2292 
               
               
                 9 
                 HBSS/f-MLP 
                 1097 
                 1775 
                 2185 
                 2411 
                 2432 
               
               
                 10 
                 Blank 
                  178 
                 187 
                 196 
                 191 
                 190 
               
               
                 11 
                 f-MLP/f-MLP 
                 1049 
                 1277 
                 1413 
                 1538 
                 1582 
               
               
                 12 
                 0.3 mL cells 
                     9999 4   
                 9999 
                 9999 
                 9999 
                 9999 
               
               
                 13 
                 HBSS/HBSS 5   
                 1425 
                 1491 
                 1577 
                 1682 
                 1735 
               
               
                 14 
                 HBSS/HBSS 
                 1359 
                 1454 
                 1491 
                 1551 
                 1645 
               
               
                 15 
                 HBSS/HBSS 
                 1340 
                 1386 
                 1478 
                 1582 
                 1650 
               
               
                 16 
                 Blank 
                  179 
                 172 
                 176 
                 178 
                 171 
               
               
                 17 
                 f-MLP/f-MLP 
                 1187 
                 1181 
                 1516 
                 1622 
                 1673 
               
               
                 18 
                 0.4 mL cells 
                 9999 
                 9999 
                 9999 
                 9999 
                 9999 
               
               
                 19 
                 HBSS/f-MLP 
                 1277 
                 1573 
                 1701 
                 1836 
                 1851 
               
               
                 20 
                 HBSS/f-MLP 6   
                 1228 
                 5928 
                 6063 
                 6342 
                 6504 
               
               
                 21 
                 HBSS/f-MLP 
                 1242 
                 1207 
                 1830 
                 1931 
                 1969 
               
               
                 22 
                 Blank 
                  176 
                 171 
                 169 
                 167 
                 166 
               
               
                 23 
                 f-MLP/f-MLP 
                 1231 
                 1325 
                 1454 
                 1541 
                 1604 
               
               
                 24 
                 0.4 mL cells 
                 9999 
                 9999 
                 9999 
                 9999 
                 9999 
               
               
                   
               
               
                 Notes:  
               
               
                   1 Transwell-type inserts used for wells 1-12.  
               
               
                   2 f-MLP added to cell suspension immediately before start of experiment.  
               
               
                   3 Conc. = 2 × 10 −8  M  
               
               
                   4 Fluorescence greater than measurable at selected sensitivity setting.  
               
               
                   5 Millicell-type inserts used for wells 13-24.  
               
               
                   6 Insert leaked