The overall goal of this procedure is to mimic ischemia reperfusion in sea elegance. This is accomplished by moving sea elegance to a micro tubule and washing the bacteria out. The second step is to create an anoxic environment.
Using a lab made hypoxic chamber, the animals are subjected to anoxia for 20 hours and then placed in aerobic conditions after 24 hours. The final step is to score for dead animals and score for touch response. Neuronal modifications are also observed through fluorescence microscopy.
Ultimately anoxia starvation in sea elegance is used to create the same conditions seen in ischemia, injury in superior eukaryotes in an easier and more flexible genetic model, And the manage advantages of these techniques over extincting methods of ischemic repeat fusion in cells isolate hearts is that C elegance is a very powerful genetic model and also is a transport. Organisms allow you to use several different fluids and techniques. In addition of that, we are showing you how to, instead of using an expensive hypoxia or environmental chamber to use a lab me hypoxia and environmental chamber, that will just cost a few bucks.
Generally, individual news to this matter need to be aware of some key points as, for example, purging the buffers and being very careful with scoring the worms and also being constant with the amount of temperature that is being using and the amount of oxygen. All of these points can change the outcome of the experiment To prepare a custom hypoxic chamber, a Tupperware style sealable airtight container can be retrofit as an anoxic chamber. Select the smallest possible container as a small container creates a hypoxic environment faster and is easier to fit in the water bath.
Make two holes in the lid that are sized to accommodate a plastic or metal connector that fits the tubing secure in place with glue that can be placed under water and also gets hard after drying, avoiding movement of the connector and hence reducing the possibility of leakage. Alternatively, a neoprene washer can be used to help seal the connector to the container. Place the container into the water bath so that it is submerged completely.
To immerse the container. Use a bottle weight or a diving weight. One of the tube connections will be used to introduce a gas mixture and the other tube will act as a pressure relief.
Therefore, it should be left under the water so that the container is sealed off from the external environment. To begin grow c elegance as described in the text protocol, prepare M nine buffer and purge the oxygen by equilibrating with nitrogen 30 minutes prior to the experiment and keeping surrounded by ice. Make a small hole in the lid of 1.5 milliliter micro centrifuge tubes in which the worms will be placed during anoxia starvation.
The hole allows for gas exchange without keeping the lid open, which can lead to excessive media evaporation. Add one milliliter of room temperature oxygenated M nine buffer to each 35 millimeter plate containing young adult worms. Be careful to avoid removing bacteria from the plate.
After approximately one minute, the worms should be swimming in the M nine. Next coat, A one milliliter pipette tip with bovine serum albumin or BSA by pipetting and expelling a sterile 1%BSA solution. Carefully incline the plate containing the worms in M nine buffer and remove the M nine from the same place where it was added.
Then place the suspension in the prepared micro centrifuge tubes. Let the samples rest in ice until the worms have dropped to the bottom of the tubes. Remove the maximum amount of M nine possible without disturbing the worms from the tube.
Then add one milliliter of the oxygen purged and iced M nine, and place the tube over ice until the worms settle to the bottom.Again. Repeat this washing step three times in the last wash. Remove the M nine until approximately 100 microliters remains in the tube.
Next place the tubes with the worms in Deoxygenated M nine buffer into the chamber and seal the chamber using the lid gas with a constant nitrogen flux of 100 milliliters per minute. Place the container inside a water bath at 26 degrees Celsius. Ensure that the exit tube is well immersed in water so no air returns and that there are no leaks in the system.
Proceed to incubate the worms at 26 degrees Celsius for 20 hours following incubation pipette sea elegance with a BSA coated tip onto a 35 millimeter nematode growth media plate. Incubate the plates at 20 degrees Celsius for 24 hours. After this, the worms will be ready to score.
Using a platinum pick, lightly touch the top of the head of the worm Live. Worms will move backwards after being touched if the worms are nonresponsive, score as dead. Occasionally a worm will not move backwards, but may exhibit a slight side to side head movement, although not technically dead, these worms are almost certain to expire within hours and should be scored as dead.
Repeat this step 10 to 15 times for each worm with ten second intervals. Probe at least 10 worms in each group to have accurate data to visualize Post anoxia starvation, neuronal modifications. Use a strain which expresses green fluorescent protein or GFP in the touch responding mech neurons.
Place a drop of 2%ARO solution on a glass slide that has been placed between two other slides that have a single piece of tape on their bottom side. Place a final glass slide perpendicular to the first on top of the 2%ARO solution and let it cool at room temperature. This should form a pad that is the thickness of the tape.
Then remove the top slide and add 10 microliters of M nine containing 0.1%tetra aole, which will immobilize the worms. Move about 10 live worms onto the 2%aro slide and place a cover slip over them. Visualize GFP using a fluorescence microscope under a 100 x objective with the appropriate illumination.
Damaged neurons will show a cytosolic membrane string of pearls pattern in the processes comprised of multiple punctum and or abnormalities where the processes appear to be broken. Count punctum and abnormalities in both neurons for each worm using a total of at least 10 worms. Subjecting sea elegance to 20 hours of anoxia and starvation at 26 degrees Celsius in a custom lab made incubation chamber resulted in significant mortality.
The survivors also responded poorly to light body wall touch. Subsequent fluorescent imaging of puncto and breaks in the GFP labeled neuronal processes of survivors confirmed the presence of morphological abnormalities. After watching this video, you should have a good understanding how to promote ischemia.
An observation in lab made hypoxic chambers and also you should be able to measure death and also neuro modifications in Seattle elegance. This model has been used by multiple groups to study how in natural ion from much interventions and metabolism can influence life and death earner hypoxic conditions.
