Chemotaxis chamber

It is important to examine the fluorescent conjugate for biological activity and confirm that the coupling procedure does not destroy the chemoattractant stimulatory activity. We evaluate neutrophil chemotaxis in a 48-well micro-chemotaxis chamber (7) as previously described in detail (8) and briefly outlined below. 

Pipet -25 pL of chemoattractant in each lower well of the chemotaxis chamber. 

Zicha D, Dunn GA, Brown AF (1991) A new direct-viewing chemotaxis chamber. J Cell Sci 99(Pt 4) 769-775... 

Multi well chemotaxis chamber and an accessory pack containing clips and a wiper blade (Neuro Probe AP48 and P48AP, respectively). 

Using gloves and forceps, place the polycarbonate filter over the chemokine- or medium-filled wells of the chamber. Adjust the position of the filter if the peripheral wells are not covered. Over adjustment of the filter may result in crosscontamination of the wells in the chamber. The chamber gasket and lid should be assembled quickly after the placement of the filter. Pressure should be applied to the center and the comers of the chemotaxis chamber lid prior to final chamber assembly to prevent cross-contamination and bubble formation. 

Dilute the purified cell suspension to the concentration of 1 x 106 cells/mL in chemotaxis medium. For 48-well microchemotaxis chambers, 2.5 mL of cell suspension will be required for each chamber. Add 50 pL of the cell suspension to each of the upper wells of the chemotaxis chamber. Once added, incubate in a 37°C humidified-air C02 chamber. 

Chemotaxis assays provide a very sensitive indicator of cellular mobilization and can easily be mastered by anyone with average laboratory experience. Several critical steps must be followed in loading a chemotaxis chamber. The first is to add the correct volume of chemokine to the bottom wells of the chamber. Typically, 26 pL is the correct volume to be loaded. Too small a volume results in air bubbles in the wells, while too large a volume overflows the wells. 

Put 4 x 10s cells are 28 uL of medium containing 1% BSA in each well in the lower compartment of the 48-well Boyden Chemotaxis chamber. 

Invert the chemotaxis chambers and incubate for 2 h at 37°C to allow the cells to attach to the membrane. 

Each data point should be carried out in at least triplicate. This allows for a maximum of 16 data points per chemotaxis chamber (48 wells in total). The design of the apparatus ensures that there is little cross-contamination between wells. In addition, the wells are arranged to ensure that it is easy to orientate the filter and identify individual wells. 

Amnion mounting. Use a 24-well two-compartment chemotaxis chamber apparatus, with 1.6 cm well diameter. Stretch and clamp the denuded BM, either labeled or nonlabeled, within the holders, thus dividing the upper and lower compartments (Fig. 4.3). For invasion studies, sandwich a Millipore filter (8 /xm pore size) in direct contact with the amnion stromal surface. 

Matrigel (Collaborative Res., Beckton Dickinson) blind well chemotaxis chambers with 13-mm diameter filters polyvinylpyrrolidone-free polycarbonate filters, 8 or 12 /xm pore size (depending on the cell size). 

Fisher, P.R., Merkl, R. and Gerisch, G. (1989). Quantitative analysis of cell motility and chemotaxis in Dictyostelium discoideum by using an image processing system and a novel chemotaxis chamber providing stationaiy chemical gradients. /. Cell Biol. 108, 973-984. 

Chemotaxis in Microfluidic Chemotaxis Chamber 2.1.1 Making Microfluidic Chemotaxis Chamber... 

To coat the microfluidic chemotaxis chamber, the microfluidic device is refilled with HBSS buffer and the device is viewed under an inverted microscope to verify that no air bubbles are trapped if bubbles are present. 

In a generalized Adler method, one end of a capillary tube (1 Xl disposable micropipette, 3 cm long with an internal diameter of 0.2 mm) is flame sealed. The entire capillary tube is then quickly passed through a flame, and while warm, the open end is plunged into a reservoir containing the test chemical dissolved in chemotaxis medium. The liquid is drawn up into the capillary as it cools and the filled capillary is then withdrawn from the reservoir and inserted into a chemotaxis chamber, which is constructed by placing a U-shaped melting point capillary tube between a microscope slide and a coverslip (Fig. 1.2). The chamber is filled with an appropriate chemotaxis medium and inoculated with bacteria so that the final concentration is approximately 6 x 10 cells/ml. After a 1-h incubation, the capillary is removed from the chamber and the exterior rinsed with sterile water. The sealed end of the capillary is then broken and the contents are... 

The advantages of the capillary assay are its simplicity, quantitative nature, and high sensitivity. Alternative methods for studying chemotaxis such as the swarm plate method of Adler (1966) require that the chemoattractant be metabolized. This is not necessary in the standard capillary assay. In addition, due to the small size of the chemotaxis chamber, only small amounts of compound are required to perform the experiments. The main disadvantage of this method is that the compound tested must be soluble in the chemotaxis medium. 

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... 

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