1. Introduction Interest in the role of cell adhesion molecules in complex cell-cell and cell-matrix interactions con- tinues to grow at a rapid pace. We have developed a novel, simple assay for cell adhesion [1] that we now extend to cellular chemotaxis and invasion. The present method is ideal for the identification of antag- onists or agonists of specific cell adhesion molecules that are active during chemotaxis and invasion. Measurements of cellular chemotaxis and invasion have historically been made in Boyden-type cham- bers with polycarbonate membranes coated with reconstituted basement membrane mixtures such as Matrigel  ; cells are added for a defined length of time, and those that migrate to the lower well are stained and counted. Modifications of this basic assay have been made using 24-well plates fitted with membrane inserts [2]. We have previously used such a method to test the chemotaxis and invasion of cultured metastatic and nonmetastatic murine adrenal carcinomas [3]. Although this staining approach is straightforward, it has several disadvantages: First, quantitation can be subjective and labor-intensive since cells are counted microscopically. Second, stained cells cannot be manipulated further to test for specific markers of interest. To enhance the flexibility and objectivity of this method, some investigators have prelabeled cells with compounds such as the esterase substrate, calcein, coupled to a fluorichrome. Although calcein has been shown not to affect the chemotaxis of leukocytes [4, 5], less is known about its biological effect or tendency to leak from other cell types. Described here is a quantitative, non-radioactive in vitro assay to measure cellular chemotaxis and invasion without pre-labeling of cells. It differs from other chamber assays in a number of important ways. First, fluorimetric measurements of cellular actin make quantitation of immobilized cells objective and sensitive while providing the potential for high throughput applications. Second, by labeling cells after experimental manipulation, normal biological functions necessary for the multi-step migration process are not disrupted. Third, the assay is fluori- metric rather than radiometric which permits auto- matic detection in a standard microtiter fluorimeter or by fluorescence microscopy. Fourth, further testing for specific cellular markers can be easily accom- plished by co-staining with any fluorochrome-tagged compound. This is especially advantageous when documenting the migration of mixed cell populations. Finally, the experiment is done in a disposable chamber with small volumes which conserves reagents and precludes cross contamination from previous experiments. Actin has historically been used to quantitate cellular lysates in Northern, Southern and Western blots. Here it is used for biological quantitation of whole cells. In this case, actin is tagged following Methods in Cell Science 19: 189–195 (1997)  1997 Kluwer Academic Publishers. Printed in the Netherlands. Rapid and quantitative in vitro measurement of cellular chemotaxis and invasion Margaret B. Penno 1 , Jennifer C. Hart 1 , Shaker A. Mousa 2 & Jeffrey M. Bozarth 2 1 The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; 2 The DuPont Merck Pharmaceutical Co., Wilmington, Delaware, USA Accepted in revised form 10 July 1997 Abstract. An in vitro, fluorimetric method for cellular chemotaxis and invasion has been developed using a commercially available, disposable, 96-well chamber. This 4–18 hour microtiter chamber assay has a number of important advantages over existing methods. It does not require prior labeling of cells or radioactivity, and is rapid, automatable and quan- titative. Cells are quantitated by a novel actin-based fluorescence tag as reported previously (Methods in Cell Science 17: 263–270, 1995). Following quanti- Key words: Chemotaxis, Fluorescence measurement, Integrin, Invasion, In vitro migration, Lung tumor, Metastasis, Quantitation tation, cells are easily detectable by fluorescence microscopy. In addition, this assay conserves reagents due to its low volumes in the upper and lower chambers. The assay has been optimized using cultured human lung cancer cells to identify inhibitors or activators of directed cell migration. The effects of antibodies to αVβ3, αVβ5, and CD44 on the chemotaxis and invasion of A549 cultured lung tumor cells are reported.