Polycarbonate filter assay

Faller and Wohnsland [509,554] developed the PAMPA assay using phospholipid-free hexadecane, supported on 10-pm thick polycarbonate filters(20% porosity,... 

Faller and Wohnsland [18, 19] developed the PAMPA assay using phospholipid-free hexadecane, supported on 10 pm-thick polycarbonate filters, and were able to demonstrate interesting predictions. Their PAMPA method appeared to be a satisfactory substitute for obtaining alkane/water partition coefficients, which are usually very difficult to measure directly, due to the poor solubility of drug molecules in alkanes. Apparently, membrane retention was not measured. 

Two types of filters are available polycarbonate and cellulose nitrate. Polycarbonate filters are used for endothelial chemotaxis assays. These filters are sided, with a matt and a shiny surface. The cells are allowed to adhere to the shiny surface prior to migration, and then stimulated to migrate to the matt surface. 

Optimal Amounts of Reagents. The functional purity of [ I]PA and the optimal amounts of beads and tracer to use for routine assay of fluid-phase (PA reactive) IgG are determined from the binding curves shown in Fig. 1. Increasing amounts of beads (0.1 ml 5-200 /ag of beads corresponding to 15-600 ng of rabbit IgG) are incubated for 60 min at 30° with [125IJPA (0.1 ml). The beads are washed with two 3-ml portions of buffer by centrifugation at 1500 g (4°) for 5 min or by filtration on polycarbonate filters, and the radioactivity in the bead pellets is determined. In this ex-... 

Like the cellulose filter assay, cells migrate through a filter that separates two chambers, with the cells in the top chamber and chemoattractant in the bottom. Because polycarbonate filters are very thin, migration in two dimensions along the top surface of the filter is primarily measured. This assay does not distinguish between chemokinesis and chemotaxis. 

Willey and Waterbury (1989) used a substantially different method to study chemotaxis in the marine cyanobacterium Synechococcus sp. This experiment involved the use of blind-well chemotaxis chambers (Neuroprobe, Inc., Cabin John, MD) that consisted of an upper (800- il) and lower (200- li1) acrylic chamber separated by a polycarbonate filter (3.0 pm). A cell suspension (165 pi) of the cyanobacterium was placed in the lower chamber over which the polycarbonate filter was placed. An air space was left between the cell suspension and the filter to control the starting time of the experiment. The upper chamber was filled with sterile seawater containing the compound to be tested and then inverted, allowing the cell suspension to contact the polycarbonate filter and the seawater/compound solution. The experiments were run for 65 min, after which time the chambers were inverted to stop the experiment. The number of cells crossing the filter into the seawater chamber was determined by direct cell counts using epifluorescence microscopy. The motile strain of Synechococcus sp. tested in this assay elicited positive chemotaxis to compounds such as ammonia, nitrate, urea, glycine, and P-alanine. Control chambers with the same concentration of chemoattractant in both the upper and lower chambers failed to elicit a chemotactic response. While the compounds tested in this study were relatively simple metabolites, one could... 

DeGendt, C. M. DeClerck, L. S. Bridts, C. H. Stevens, W. J. The use of calceinacetomethylester (AM)-labeled poly-morphonuclear cells in a polycarbonate filter chemotaxis assay. Clin. Chim. Acta 1996, 249, 189-195. 

In the same way, o-nitrophenyl octyl ether (o-NPOE) was immobilized on polycarbonate (PC) filters and the apparent permeability measured after 5 h incubation time was correlated to log Pnpoe for a series of reference compounds (log Pnpoe ranging from —1 to 3.6) [90]. Lipophilicity values in the alkane/water system were also determined using PAMPA with hexadecane-PC coated filters [89]. In this case, a correlation was found between intrinsic permeability (log Pq, permeability corrected for ionization and for unstirred water layer contribution, which particularly affects permeability of lipophilic compounds) and log P ik. However, log Pq is obtained from the knowledge of the pJC, value(s) and the permeability pH profile and therefore requires the full permeability pH profile to be measured for each compound, which negatively impacts the assay throughput. 

Multi-well modified Boyden chambers can be obtained from Neuro Probe Inc. (Gaithersburg, MD). Model AP48 has been widely used for endothelial cell chemotaxis assays. The apparatus consists of top and bottom acrylic plates, a silicon gasket and assembly screws. The bottom plate has 48 wells, each with 25 pL final volume. These correspond to holes on the top plate, and form the upper wells when the chamber is assembled. The filter (polycarbonate, 25 x 80 mm) is placed between the top and bottom plates, and a gasket is placed over the filter to create the seal. The apparatus can be purchased with a selection of accessories, such as curved forceps, filter clamps, and wipers. These are required to process the filters after use. Filters can also be obtained from Nucleopore Inc. (Pleasanton, CA) and Costar (Cambridge, MA). 

Horwitz, D. A. and Garrett, M. A. (1971). Use of leukocyte chemotaxis in vitro to assay mediators generated by immune reactions. I. Quantitation of mononuclear and polymorphonuclear leukocyte chemotaxis with polycarbonate (nuclepore) filters. J. Immunol. 106, 649-655. 

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