Chemotaxis procedures

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. 

Two bioassay techniques, the capillary method [58], and the drop method [63], have already been developed to study the chemotaxis of phytopathogenic zoospores and gametes of marine brown algae. We have devised a new procedure (the "particle method") which is a qualitative, rather than a quantitative, method of detecting an attractant after it has been absorbed on to an inorganic particle. This method has been used to study attractants of A. cochlioides zoospores in the roots of both spinach and pigweed (Chenopodium album). 

Zebrafish have emerged as a powerfiil model organism to study neutrophil chemotaxis and inflammation in vivo. Studies of neutrophil chemotaxis in animal models have previously been hampered both by the limited number of specimens available for analysis and by the need for invasive procedures to perform intravital microscopy. Due to the transparency and cell permeability of zebrafish embryos these limitations are circumvented, and the zebrafish system is amenable to both live time-lapse imaging of neutrophil chemotaxis and for screening of the effects of chemical compounds on the inflammatory response in vivo. Here, we describe methods to analyze neutrophil-directed migration toward wounds using both fixed embryos by myeloperoxidase activity assay, and live embryos by time-lapse microscopy. Further, methods are described for the evaluation of the effects of chemical compounds on neutrophil motility and the innate immune responses in zebrafish embryos. 

This is optional procedure, and the purpose is to inhibit ade-nylyl cyclase and reduce any basal chemotaxis signaling. The treatment also can be done with 1 mM caffeine for 30 min. We also point out that caffeine potentially inhibits many other cellular activities, including P13K and TOR kinase activity (36, 37). Thus one should consider the use of caffeine according to the aim of the experiment. Also one can examine the side effect of the caffeine treatment by using ACA null strain. 

In view of the notion that chemotaxis is required for the selection of capacitated spermatozoa, faulty precontact sperm-egg communication may be one of the causes of male infertility, female infertility, or both. It is reasonable that, in the future, chemotaxis may be exploited as a diagnostic tool for sperm quality and male infertility and may be used as a biological sperm-selection procedure before in vitro fertilization, especially before intracytoplasmic single sperm insertion or for intrauterine insemination. Moreover, the chemotactic activity of a follicular fluid may be an effective way to monitor the possible effects of different ovulation induction protocols on the maturational status of oocytes aspirated for in vitro fertilization [2]. On the other hand, interference vwth sperm chemotaxis may represent an exciting new approach to contraception. 

---