This video will demonstrate how to obtain hemocytes (blood cells) from the Hawaiian bobtail squid, Euprymna scolopes for use in cell biological and bacterial adhesion assays. Hemocytes will be stained with a fluorescent dye and exposed to GFP-labeled bacteria.
Representative Results
Because cephalopod hemolymph contains extracellular hemocyanin and not hemoglobin, upon oxygenation, the hemolymph will turn dark blue. An average of ~5000 hemocytes per μl of hemolymph will be obtained using this method. After adherence to the chambered cover slips and fluorescent staining, the hemocytes should appear brightly fluorescently red and amoeboid in shape. For bacterial adhesion, V. fischeri will adhere poorly to the hemocytes (1-2 bacterial cells per blood cell) while V. harveyi will adhere strongly (10-15 bacterial cells per hemocyte).
Figure 1. Adult Hawaiian bobtail squid Euprymna scolopes showing position of cephalic blood vessel.
Figure 2. Results of hemocyte exposure to Vibrio fischeri (A) and Vibrio harveyi (B). Red, Cell Tracker Orange; Green, GFP-labeled bacteria.
Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made significant contributions to our understanding of the co-evolution of eukaryotes with their microbiota. The squid/vibrio system has proven itself as a tractable model system to answer fundamental questions in this field2,3,5,6,8. The light-organ of the squid Euprymna scolopes permits colonization exclusively by the luminous bacterium Vibrio fischeri. Because the tissues that house the bacteria remain in contact with seawater, the squid must not only foster the specific symbiosis but also continue to exclude other bacteria. Continued studies have revealed that macrophage-like hemocytes likely play an important role in the establishment and maintenance of this association1,4,7. Because the squid host lacks adaptive immunity, the amazing specificity found in this association must be whole or partially mediated through the innate immune system. A recent investigation of these blood cells revealed that hemocytes isolated from E. scolopes recognize and phagocytose V. fischeri and non-symbiotic bacteria differentially and that colonization likely leads to a type of “immune tolerance” of the symbionts4. This protocol will demonstrate how to successfully obtain these blood cells from adult squid and test their ability to bind bacteria.
Funding sources: University of Connecticut Research Foundation and the Department of Molecular and Cell Biology to SVN, Sigma Xi Grant-in-Aid-of Research and Antonio H. and Majorie R.
Romano Graduate Education Fellowship to AJC
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
Lab-Tek chambered #1.0 borosilicate coverglass system (8-chambers) | Thermo-Fisher Scientific | 155411 | ||
26.5 G 1ml latex-free insulin syringe | Becton, Dickinson and Company | C34551 | ||
Cell Tracker Orange | Invitrogen | C34551 | ||
SteREO Discovery V20 Microscope | Zeiss | |||
SP2 Confocal Microscope | Leica |