The overall goal of the following experiment is to evaluate sleep and immune function in drosophila. This is first achieved by recording locomotor activity of infected flies to measure sleep and survival as a second step after infection flies are homogenized, diluted, and spread onto LB agar plates. The number of colony forming units that grow indicates the ability of flies to clear the infection.
Next infected flies expressing a transgene containing the NF kappa b luciferase reporter are added to a 96 well plate containing luciferian, the substrate of luciferase. Real-time measurement of kappa b Luciferase indicates activation of an NF kappa B signaling pathway during infection altogether sleep survival outcome. Bacterial clearance and NF KB reporter activity are measurements that provide mechanistic insight into the molecular link between immune function and behavior.
Generally, individuals new to this method or struggle because it takes time and practice to learn how to manually infect flies with consistency. The main advantage of the Lucifer reported technique over existing methods such as QPCR is that NF Kappa B activity can be measured continuously in real time in living. Flies Begin by incubating late pupil staged drosophila cultures for three to four days, so the adults adapt to environmental conditions.
Here the flies are placed under constant light to eliminate the influence of the circadian clock on the immune response and behavior. Next load one to four day old flies into the activity. Monitors record their activity for a minimum of three days prior to the infection one day before the scheduled infection.
Pick a single SIA colony with a sterile pipette tip and submerge the tip into a culture tube containing five milliliters of LB medium to verify sterile technique. Do not forget to make a control mock culture without bacteria. Grow bacteria overnight until it reaches the exponential growth phase The next day.
Measure the culture's absorbance at 600 nanometers. Include a second Q vet as a blank. It is ready when the concentration is between 0.5 to one absorbance units subculture, or extend the culture time as needed.
Now prepare the bacterial solution to infect the flies. Dilute the bacteria with PBS to an expected absorbance of 0.1 at 600 nanometers. Then add food coloring for the injection control.
Replace the bacteria with LB medium. Store both solutions on ice. Next, using a micro pipette puller, make injection needles with a fine tip under a dissecting microscope.
Use fine forceps to break off the tip of each needle so that the opening is large enough to fill with injection fluid using suction, but also small enough to minimize damage to the fly. Attach a glass needle to a three cc plastic syringe with a length of tubing using the syringe. The flow of injection medium is controlled manually.
Avoid contaminating the rubber tubing with injection fluid as the syringe apparatus is used for both infective and control injection. Verify the flow of injection medium with the use of a Kim wipe tissue. Two critical details to successful injections are using the optimal needle size and the secure seal between the needle and the syringe.
Once the injection system is fully prepared, anesthetize the flies using a minimal flow of carbon dioxide, proceed swiftly so no flies anesthetized for longer than five minutes. Now inject flies by poking the glass needle into the region above the skew tellum of the dorsal thorax. The passing of injection medium into the fly is verified by the food coloring, which can be seen as the injected solution spreads.
Before beginning, make certain that all the materials used for this procedure have been autoclave also at least a day in advance. Prepare 10 centimeter petri dishes with LV agar medium. On the day of the experiment, anesthetize and place flies into 1.5 milliliter centrifuge tubes.
Prepare a minimum of two groups of 10 flies each per experimental condition. Also prepare a control group of flies without infection, especially when using a bacterial strain without antibiotic resistance. Store all the loaded tubes on ice.
Next, add 400 microliters of LB medium to each fly loaded tube. Homogenize the flies using a small peal and do this near a flame to prevent contamination. Now using the cut pipette tips, serial dilute the homogenate by factors of 10 in volumes of 200 microliters.
If the homogenate is made immediately post infection with sia, make dilu up to one to 1000, but for 24 hours, post infection homogenate, make up to one to 100, 000. Next seed, the largest two dilu on 10 centimeter plates using glass balls to ensure an even distribution, incubate the plates overnight. The next day, count the numbers of colonies on the plate by direct observation or via colony counting software.
Then calculate the number of colony forming units per fly. This assay uses transgenic flies carrying the kappa b luciferase reporter at the appropriate time. The assay also requires cultured bacteria such as SCIA prepared for injection as described in the previous section.
Adjust the flies to the experimental lighting conditions. In this example, one to four day old kappa b luciferase flies are housed in vials containing 5%sucrose, 2%agar food medium, and placed in constant light for two days. Now prepare a 96 Well microplate for the flies.
Fill each well with two layers of food. Medium first, add 300 microliters of 5%sucrose, 2%agar solution to each. Well cover the plate with a fine mesh cloth and allow the plate to dry thoroughly for up to an hour.
Next, add a 50 microliter top layer to each well containing 5%sucrose, 1%agar, and two millimolar Lucifer. Because Lucifer is light sensitive, allow the plate to dry in the dark and going forward. Minimize the plate's light exposure.
Once the agar has cooled, cover the plate with clear adhesive film. Then using a fine needle, perforate the film twice over each well. These holes allow air exchange and gaseous anesthesia.
Next, to facilitate fly loading, use a sharp blade and straight edge to introduce a cut between each column. Anesthetize the flies with carbon dioxide and load them one by one to each well column by column. Should a fly get stuck to the film, give it an opportunity to free itself before intervening.
Return the micro well plate to the incubator under constant light for eight to 24 hours. To anesthetize the well bound flies, use a micro pipette tip attached to a low pressure carbon dioxide line. Be sure the gas is at harmlessly low pressure and place the micro pipette tip directly above the ventilation holes to anesthetize a fly in groups of eight individually.
Transfer each fly to a CO2 pad and infect them with bacteria as previously. Then return each fly to its original well and reseal the microplate. Measure the luminescence in each fly using a luminometer housed in a temperature controlled room under a defined lighting schedule.
Load the plates into the stacking cassette, making sure to stack the plates containing the flies between clear blank plates. Program the luminometer according to the manufacturer's specifications and collect readings every hour for a minimum of 24 hours. In this example, the detectors are programmed to read each well for 10 seconds.
Once completed, export the data files to a spreadsheet and perform a standard analysis graphing the results. The example data can be analyzed. Discounts per second averaged from three readings per well.
Canton S wild type flies and relish. E 20 mutant flies lacking in NF kappa B gene were loaded into two activity monitors in constant light and infected with sia. In this example, infection promoted sleep.
It was clear that relish E 20 mutants experienced less sleep than Canton s. Control flies after infection. Consistent with previous findings, the relish E 20 mutants also rapidly succumbed to the infection as compared to wild type flies.
Most flies survived the aseptic control injection indicating the flies succumbed to the infection and not to the injury due to the injection. Because the relish mutants died so quickly, only Canton s flies were used to demonstrate bacterial load after infection. Sia continued to proliferate in the fly 24 hours after infection.
Lucifer's reporter activity showed large variation between individual flies and between success of data points. Although the signal is derived from all tissues within the fly, it is not expected that every tissue would emit the same amount of signal and the visibility of the tissue to the detector would vary as the fly moves. In this example, flies were infected and compared with a handled control group.
Flies that died were excluded from the analysis across a group of flies. Kappa b Luciferase reporter activity steadily rises after infection. The activity of the Kappa b Luciferase reporter also rose steadily after aseptic injury.
Peak activity was around 12 hours for both post-injury and post-infection. After watching this video, you should have a good understanding of how to quantify immune function and sleep in SSO after infection. We can measure sleep, survival, time, bacterial clearance, and the real time activity of an un copy transcription reporter.
