Chemotaxis Volume

Volume 162. Immunochemical Techniques (Part L Chemotaxis and Inflammation)... 

Nitrous oxide decreases tidal volume and increases the rate of breathing and minute ventilation. Although arterial carbon dioxide partial pressures tend not to be affected the normal ventilatory responses to carbon dioxide and to hypoxia are depressed. Alveolar collapse in structured lung segments may be more rapid in the presence of nitrous oxide than with oxygen due to its greater solubility. Similarly, it depresses mucous flow and chemotaxis. In theory these factors predispose to postoperative respiratoiy complications. 

Chemotaxis experiments (see Note 2) Use blind well Boyden chambers, that contain a volume of 100 pL in the lower compartment (Costar, Bodenheim, Germany). As filters use selfpunched (7 pm punch) polyvinylpyrrolidone containing polycarbonate filters (pore size 3 mm, Costar), which prior to use need to be washed with 1 M NaOH in 50 % (v/v) aqueous ethanol for 7 min followed by three washes in water (see Note 3). Use p-nitrophenyl-p-D-glucuronide (Sigma) at 10 mM in 0.1 M aqueous sodium acetate, pH 4.0, for p-glucuronidase detection (see Note 4). 

Up to 1% of the fraction volume in the assay system will be tolerated without affecting chemotaxis. When higher percentages need to be used these should be lyophilized in the presence of BSAcontaining PBS prior to bioassay. 

Functional assays such as Ca2+ mobilization and chemotaxis (see Chapters 16 and 11 respectively, in this volume) or microphysiometer analysis (4) in response to ligand stimulation. 

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. 

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). 

Like other NSAIDs, the drug inhibits prostaglandin (PG) synthesis chemotaxis and the release of liposomal enzymes (from liver). It has been observed duly that a chronic administration with 20mg per day causes steady state plasma levels of 3-5 meg. mL within a span of 7 to 12 days. The volume of... 

One such application is to observe and quantify the morphogenesis and cell migration through the matrix scaffold. For instance, a microfiuidic platform named KOALA has been reported to enable neutrophil purification and investigate neutrophil chemotaxis simultaneously to better understand the mechanisms of neutrophil recruitment [1]. This microfiuidic platform allowed the investigation of neutrophil chemotaxis to be carried out with low sample volume (e.g., nanoliters) and estabfished a gradient of chemotactic agents to direct the... 

---