Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

After bobtail squid adults have been removed from their tanks and anesthetized with a 2%ethanol filtered seawater solution. Their bled through the cephalic blood vessel. With a 26 and a half gauge syringe, the extracted hemolymph appears blue.

Due to the presence of hemo cyan, the he cytes are diluted to a desired concentration and aliquot into an eight chambered cover slip. Once the he cytes have adhered to the cover slip, they're stained with a cell tracker orange, a fluorescent dye, and challenged with GFP labeled bacteria, which can be visualized on a fluorescent microscope. Hi, I am Spencer Olm in the Department of Molecular and Cell Biology at the University of Connecticut.

And I'm Andrew Collins from the NY Home Lab. Today we will show you a procedure for extracting hemolymph or blood from the Hawaiian bobtail squid. You prime this opis.

Now bobtail squid are nocturnal animals found off the coast of Hawaii near coral reefs, and they use the light produced by their symbiance to camouflage themselves at night when they're out hunting for their favorite food, which is shrimp. Now we use this association in the lab to study how beneficial bacteria colonize animal tissues. We use this procedure in our laboratory to study interactions between components of the innate immune system of the host and its bioluminescent bacterial partner VIO ide.

So let's get started. Before extracting Hemolymph anesthetize one adult squid in a one liter beaker containing a freshly prepared solution of 2%ethanol in 500 milliliters of filter sterilized artificial sea water. After 10 minutes, the squid will see swimming and will not actively respond to touch.

However, continued respiration as indicated by movement of the mantle and chromatophore activity indicating activity of the pigment cells should still be observed. Pour some of the sterile seawater ethanol solution from the beaker into a standard wax dissection tray and submerge the squid in the tray with the ventral site up. Using one standard 200 microliter pipette tip, pull back the funnel and mantle to expose the main cephalic blood vessel located between the two eyes.

Use a sterile syringe with a 26 and a half gauge needle to puncture the cephalic blood vessel and withdraw between 50 to 100 microliters of hemolymph or 10 to 20 microliters. If the animal will serve as a donor multiple times, place the hemolymph in a sterile 1.5 milliliter tube on ice. Note that if this step is done correctly, the squid blood should appear dark blue.

Replace the animal in a normal seawater tank to revive spin the hemolymph in a micro fuge at room temperature to pellet the hemo and pipe it off the supernatant. Re suspend the hemos in 500 microliters of squid ringer solution to wash. Then spin us before to pellet the cells and remove the supernatant.

Finally, resuspend the hemos and squid ringer solution. Determine cell concentration by hemo cytometer. Then add approximately 2000 cells to each chamber of a glass cover slip.

Allow the cells to adhere to the glass for approximately 10 minutes. At this density, the hemos should form a uniform monolayer on the glass surface. Keep the hemos on ice while preparing bacteria in an orbital shaker at 28 degrees Celsius.

Grow one or more strains of Vireo bacteria containing a green fluorescent reporter gene to mid log phase in seawater tritone medium. Using a spectrophotometer measure the optical density of the culture at a 600 to determine cell density in a centrifuge pellet the bacteria discard the snat and resuspend the pellet. In one milliliter of squid ringer solution, add bacteria to the hemo cytes.

In the chambered glass slides with 100, 000 bacteria per chamber. Incubate the slides at room temperature for one hour. To achieve maximum binding add cell tracker orange, add a final concentration of 0.005%to stain the hemo cytoplasm.

Incubate for 30 minutes and then wash with 150 microliters of fresh squid ringer solution. Add a confocal microscope, view the stained he cytes and associated bacteria enumerate the bacteria over the entire surface of the he cyte. Now we'll show you some representative images of he cytes challenged with the GFP labeled non symbiotic bacterium Filio Harvey Eye and the natural squid Symbio Filio fii.

Here is an image of a hemo site that was not challenged with any bacteria. The cell appears red and has become activated. So you can see its pseudopodia like extensions.

When the cell is challenged with the bacterial symbio filio fii, a few bacteria are seen binding to the surface of the cell. However, when the non symbiotic bacteria filio Harvey Harveyi is exposed to the hemos, significantly more bacteria are bound. We've just shown you how to extract hemo lymphs from a bobtail squid, isolate the hemos and use them in experiments to characterize bacterial adhesion to these squid cells.

This protocol could be modified for immunochemistry by fixing the hemo cytes after they adhere to the cover slip surface. R-N-A-D-N-A and proteins may also be extracted once the hemo cytes have been obtained from the animal. So that's it.

Thanks for watching and good luck with your experiments.