Filter assays

C2. Capsoni, F., Minonzio, F., Ongari, A. M., and Zanussi, C., A new simplified single-filter assay for Lin vitro evaluation of chemotaxis of 51Cr-labeled polymorphonuclear leukocytes. J. Immunol. Meth. 120,125-131 (1989). 

The results are expressed as the number of cells per high power field for each 10 pm distance. Statistical analysis for thick filter assays can be performed using many methods including a twotailed student to test or a KruskalWallis nonparametric analysis of variance on averaged triplicate values. 

Checkerboard analysis. The filter assay does not allow one to directly observe migration of cells, and track them to establish... 

Researchers with a knack for bargaining can achieve substantial price reductions. However, why invest so much money in a device when you can just as well determine n and Kd with an inexpensive filter assay Because of AH With AH you gain access to the anatomy of the binding. This can answer questions such as the following. 

As for the binding assay if a radioactive ligand is available, try the filter assay first. It s the fastest and its applicability usually only depends on whether or not the ligand sticks to the filter. You should be able to answer this question after a few experiments. If the filter test does not work, use the column assay. If that is not possible (e.g., because ligand and binding protein are about equal in size), you can employ PEG precipitation or the methods from Petrenko et al. (1990) or Scheer and Meldolesi (1985). 

For filter assays, you may try to wash the filters better. During washing it is not so much the volume that is important but the number of washing steps. It is better to wash three times with 4 ml than one time with 12 ml. 

Note with filter assays glass fiber filters with adsorbed or Ca must be incubated with scintillator for at least 18 h, with occasional shaking. Only then the cpm/vial will have stabilized. Preincubation of the filters with a solution of 10 mM CaCl2 should inhibit the nonspecific binding of to the filters. 

Cellulose filter assay Cell migration occurs through a porous filter... 

This assay is a variation of the cellulose filter assay that uses multiple arrangements of chemoattractant above and below the filter to distinguish chemokinesis and chemotaxis. In addition, the depth to which the cells penetrate the filter is determined. These features allow calculation of population parameters x, the random motility coefficient and X, a chemotaxis coefficient. 

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. 

Filter assays have been very popular for identifying soluble compounds that stimulate locomotion (reviewed in [424, 426]). Two chambers are separated by a filter with pore sizes less than the diameter of the cells, such that cells must actively migrate to get through. The apparatus is oriented such that cells are put in the top chamber and can settle onto the filter. Solution with chemoattractant is put in the bottom chamber, and as it diffuses through the pores a concentration gradient is established such that the cells crawl through the filter. There are two main types of filter... 

Buettner, H.M., Lauffenburger, D.A., and ZSgmond, S.H. (1989). Measurement of leukocyte motility and chemotaxis parameters with the MiUipore filter assay. /. Immunol. Meth. 123, 25-37. 

Fig.4 Cross-reactivity pattern of purified chi a/b proteins. A) Coomassie stained gel B—G) filters assayed with polyclonal antibodies to individual chi a/b proteins.

Fig. 1. (left) Effect of poly L-lysine, L-arginine or both on incoiporadon from [adenylate-32p] NAD into proteins of SR vesicles. The reaction mixture containing 0.88 mg/ml of SR and 0.5 mM labeled NAD (2 ci/mol) in the absence and presence of 50 mM L-arginine was incubated, with or without 100 [ig/ml of poly L-lysine, at 25 C for 30 min, and radioactivity of the acid-insoluble fraction was measured by filter assay (2). The minute amount of [32p]NAD which bound to poly L-lysine during the incubation was detected. Thus, this radioactivity was subtracted fiom the value obtained in the presence of SR plus poly L-lysine. The ADP-ribosylation of L-arginine was determined by HPLC analysis (6). 

Galactosidase activity was determined by a colony-lift filter assay for the 3-day-old SFY526 yeast transformants containing the indicated plasmids. 

The transformed cells are plated on medium lacking tryptophan and leucine, and subjected to a filter assay for, 3-galactosidase activity. 

For the p-galactosidase filter assay, a stock solution of 5-bromo-4-chloro-3-indolyl P-D-galactopyranoside (X-gal) is prepared fresh in dimethylformamide at a concentration of 40 mg/mL This stock solution is added with constant stir-nng to the IX Z-buffer to yield a final concentration of 0 33 mg/mL... 

After 4 d of growth at 30°C, the colonies that grow under histidine selection are assayed for lacZ expression using the -galactosidase filter assay described in Subheading 3.3.2. This protocol uses filter-paper circles that are 145 mm in diameter and these are incubated on 9 mL of Z-buffer containing X-gal dispensed in the lids of 150-mm culture dishes. 

A direct comparison of Whatman 50 filter papers v nitrocellulose filters demonstrated that the filter paper was far superior m this colony-lift filter assay The signal was more intense and developed more rapidly for colonies on the filter paper compared to colonies on the nitrocellulose filter. It is possible that substrate access may partially explain this difference. Moreover, the colonies appear to adhere better to the Whatman 50 filter paper and it is considerably less expensive than its nitrocellulose counterpart... 

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