The overall goal of this procedure is to extract, visualize, and count the he cytes from wild caterpillars. This is accomplished by first anesthetizing the caterpillar by placing it on ice, injecting it with a buffer solution containing an anticoagulant and returning the caterpillar to the ice to allow the he cytes to reenter circulation before extraction. The second step is to make a shallow incision at the posterior end of the caterpillar using a clean scalpel.
Next, the caterpillar is gently squeezed using soft forceps. Then the buffer solution hemo lymph mix is aspirated using a micro pipette and collected in a micro centrifuge tube. The final step is to gently mix the hemo lymph solution with an equal volume of buffer solution and transfer 10 microliters of the solution onto a hemo cytometer to visualize and count the number of he cytes.
Ultimately, this hemo cyte extraction method can be used as a quick and effective method to count he cytes from various caterpillar species and conserve as a tool to examine physiological aspects of the insects cellular immune response. The main advantage of this technique is that it can be used to extract hemo sots from caterpillars with very reduced prox. Other caterpillars models are bled by cutting one of the pros, unex extracting he cytes from the liquid that comes out.
In addition, this technique can be used to extract hemotype from other insect species. This method can be used to answer key questions in the field of eco immunology, such as how the insect cellular immune response varies with diet quality, developmental stage, or exposure to different pathogens. To begin this procedure, anesthetize the penultimate instar larvae by chilling them on ice for 20 minutes prior to hemo cyte extraction.
After that, fit the blunt end of a clean 16 gauge capillary needle into the tubing that is connected to a 20 milliliter glass syringe. Leave the tube containing the collection solution open on the lab bench. Slowly fill the capillary with approximately 10 to 15 microliters buffer.
Then rest the needle on a ball of clay that is placed next to the dissecting microscope. Place the caterpillar in a Petri dish. The insect should be positioned on its side so that the sphericals are clearly visible.
While viewing under a dissecting microscope, hold the caterpillar in place with a pair of forceps and place the needle close to one of the sphericals where the insect cuticle is generally softer. Then gently press the capillary needle against the body of the insect. Once the needle has penetrated the cuticle inject the total volume of solution into the caterpillar.
The caterpillar's body will swell following the injection, but it should not burst or ooze. Return the caterpillar to the iced Petri dish. Position the insect dorsally to prevent the buffer from leaking out of the wound.
Repeat the injections for each caterpillar used for hemo limp extraction. Then allow all insects to chill on ice for 30 minutes. Now, place the caterpillar in a Petri dish under the dissecting microscope.
Carefully make a shallow incision about two to three millimeters long in the posterior region of the caterpillar, using a sterile scalpel without damaging the gut or other internal organs. Next, squeeze the caterpillar gently using a pair of flexible plastic forceps while collecting approximately 10 to 20 microliters of the hemo limp mix with a 10 microliter pipette tip. Again, be careful not to injure the internal tissues.
After that, discard the caterpillars remains. Collect the hemo limp from each caterpillar in separate labeled Siliconized, 0.5 milliliter micro centrifuge tubes. Then add approximately 10 to 20 microliters of the incubation solution to each hemo limp sample collected from an individual caterpillar.
Keep the hemolymph samples on ice at all times in this step. Mix the hemolymph sample and the incubation solution immediately prior to counting. He cytes.
This treatment allows for proper separation of he cytes and prevents clumping. Use a clean pipette tip to transfer 10 microliters of hemo lymph sample mixed with the incubation solution onto a hemo cytometer. Then using a compound microscope count and record the number of he cytes in at least 15 of the large squares of the central grid of the hemo cytometer.
Add these values to obtain a total sum of the number of hemo cytes in the 15 squares. Next, rinse the hemo cytometer and its cover slide thoroughly using a wash bottle with distilled and deionized water. Then wipe it dry with Kim wipes.
Repeat the procedures for the rest of the hemo limp samples and calculate the mean number of he cytes per milliliter of hemo limp volume for each caterpillar. The brightfield microscopy image shows the extracted he cytes from nucle hin ai caterpillars at a 20 x magnification. Following this procedure, hemo cytes collected could be used to study cellular immune response and set up assays such as phagocytosis and cap, select encapsulation and pH oxidase activity.
In addition, R-N-A-D-N-A and protein can be collected unused for further assays in order to study these cellular immune response in insects. After watching this video, you should have a good understanding of how to inject caterpillars for the purpose of extracting hemolymph and how to collect hemolymph containing live hemo cytes.
