The overall goal of this procedure is to measure the effects of injected insulin on hemo lymph glucose levels in adult drosophila melan gaster. This is accomplished by first allowing starved flies to ingest a concentrated glucose solution. The second step of the procedure is to inject glucose fed flies with insulin.
The third step of the procedure is to collect an uncontaminated hemolymph sample. The final step of the procedure is to determine the hemo lymph glucose concentration. Ultimately, results can be obtained that show the exogenous insulin facilitates glucose clearance in Esof la Melan gaster through spectrometry.
The main advantage of this technique over existing methods like decapitation bleeding of adults, is that this procedure minimizes gut content. Contamination of the Hema lymph sample Collect flies within 24 hours of occlusion by anesthetizing them using humidified carbon dioxide. Sort out female flies and place them into vials containing standard diet.
Allow the flies to age for 10 days. After 10 days, transfer the flies to vials containing a five milliliter plug of 2%aros and starve them for 12 to 16 hours. Transfer the starved flies to vials containing 10%glucose soaked filter paper for one hour prior to insulin injection to make the insulin solution for injection dissolve bovine insulin in phosphate buffered saline to a final concentration of 0.01 milligrams per milliliter.
Also prepare a control solution containing only PBS. Add blue food coloring to both the insulin and control solutions to a final concentration of 0.5%and keep both solutions on ice throughout the entire procedure. Blunt the tips of freshly pulled glass needles by pressing them through a Kim wipe.
In order to create a stout tip with a larger poor diameter place the blunted needles tip up into a micro fuge tube containing either the insulin or the control solution and allow capillary action to backfill the needles. Check each needle for bubbles under a stereo microscope and discard any that have not filled cleanly. Insert the filled needles into the manual micro injector needle holder and position the holder with a microm manipulator so that the needle tip is visible through a stereo microscope.
Apply positive pressure on the fluid column in the needle by turning the micrometer knob attached to the plunger on the micro injector syringe. Touch a chem wipe to the needle tip to verify sufficient pressure for fluid flow. Once the needle has been prepared for injection a fix a calibration chart to the needle shaft with clear tape and bring the needle tip into focus through a stereo microscope under 20 x magnification.
Briefly anesthetize the glucose fed flies with humidified carbon dioxide and place them on a pad on ice for continued anesthetization at the stereo microscope. Pick up a fly with a pair of fine forceps and hold it close to the needle such that the tip is adjacent to the anterior region of the left side of the fly. Thorax bring both the needle's tip and the fly's thorax into focus.
Gently move the fly toward the needle tip so that the needle tip touches the center of the protuberance of the left presum region of the fly. Thorax continue to advance the fly onto the needle so that the needle impales the center of the presum. Apply positive pressure by advancing the syringe plunger with the micro injector's, micro manipulator knob until 0.1.
Microliter of either insulin solution or the control solution has been injected into the fly. The injection will impart a blue color to the left side of the anterior thorax. Allow the injected flies to recover in 2%Aros files in replicate groups for zero, 15 and 30 minutes before hemolymph extraction a fix a piece of double-sided tape to the carbon dioxide pad for the time.
Zero group anesthetize flies and arrange them on the tape dorsal side down in even rows with their head capsules aligned. Once the flies are fixed to the tape, grasp a flies pro bois with fine forceps and gently pull it towards the ventral and then posterior region so that the front of the head capsule is visible. While holding the probos in position, puncture the head capsule above the lenal suture using a sharp tungsten needle, being careful not to pierce entirely through the head.
After all the flies have been punctured, use a trimmed wooden applicator stick to apply gentle pressure to the fly's abdomen. This will produce a small droplet of hemolymph at the head capsule puncture site. Touch the end of a one microliter micro capillary tube to the hemo lymph droplet.
Determine and record the amount of hemo lymph collected by checking the fluid column within the micro capillary tube against a graduated volume chart using a spectrophotometer, detect the absorbance at 340 nanometers and determine the glucose concentration while type flies injected with the bovine insulin solution showed decreased hamo lymph glucose levels at the 15 and 30 minute post-injection time points relative to the control solution injected flies. After watching this video, you should have a good understanding of how to inject small volumes of solution into adult drosophila. How to extract hemolymph from adult drosophila and how to determine hemolymph glucose concentration from adult drosophila following this procedure.
Other experiments like protein concentration, determination, and hemo lymph triglyceride concentration determination can be conducted to answer questions such as, how does insulin affect related aspects of nutrient metabolism in adult drosophila?
