Monocytes and neutrophils are activated by and respond by moving toward molecules termed chemotactic factors, including the complement component C5a, fMLP, LTB4, PAF, the neuropeptide substance P, and the phorbol ester phorbol myristate acetate (PMA); phagocytic particles; and substances released by microorganisms such as fungi, bacteria, and viruses. The magnitude and time of response depends on the agonist used, but for most of these, the response time is in minutes rather than hours. Interaction of the cell with these agonists may be investigated through the analysis of the binding of fluorochrome-labeled molecules or by determining features of cellular activation such as cytokine production, surface antigen changes, shape change, or metabolic burst.
Flow cytometry may be used to count or phenotype cells before and after migration through human umbilical vein endothelial cells (HUVECS) grown on membranes. In this assay, the HUVECs are suspended in transwell tissue-culture inserts that are placed into wells in microwell plates. Medium is placed in the well below the insert. Cells are added to the wells and allowed to migrate. The nonmigrated and migrated cells are collected and counted by flow cytometry (44). The HUVECS may be pretreated with TNF, the migrating cells may be stimulated with agonists, and chemoattractants may be placed in the wells.
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