The aim of the following experiment is to set up and maintain a hypoxia chamber using a continuous flow method. This is achieved by obtaining and fabricating the necessary tubes, flasks, and chambers for the procedure. As a second step, connect the oxygen tank and select the flow rate to create the desired level of hypoxia.
Hydrate the gas by passing it through a bubble flask filled with deionized water. Connect the hydrated gas to the hypoxia chamber and place the test samples in the chamber to expose them to hypoxic conditions. Results are obtained that show Hi F1 dependent embryonic lethality in hypoxia based on observing viability to adulthood.
The main advantage of this technique over existing methods, such as glove boxes, is it's easy and inexpensive to fabricate in lab and provides an accurate and reproducible concentration of gases in the environmental chamber. While this method provides insight into the hypoxic response in the nematode C elegance, it can also be adapted to other systems, including larger model organisms and cell culture. Additionally, it can be used to study other gases, including carbon dioxide and hydrogen sulfide.
This protocol demonstrates how to construct a hypoxia chamber. Gas is stored in compressed gas tanks with defined oxygen concentrations, and a two stage regulator is attached. Gas enters the bottom of the flow tube, exiting the top at the correct flow rate.
Gas then flows into the bubble flask hydrating the gas. Hydro gas then passes into the hypoxia chamber at the inflow valve, exposing the samples to hypoxia. The gas finally vents into the room through an exhaust hole drilled in the chamber.
A Pyrex crystallization dish or in an arrow pack box can be used as the body of the environmental chamber. The volume of the container should be kept to a minimum and the material must be impermeable to gas. Drill a hole in one side of the container with a carbon or diamond tip, drill and fit with a plastic hose barb fitting.
Mix the epoxy before application to the box and fitting. Then repeat the same procedure on the opposite side of the container with the second hole offset from the first. To increase turbulent mixing, use one eighth inch outer diameter, FEP or nylon tubing to connect the compressed gas tanks to a flow control device, such as a mass flow controller or a rotor meter.
The gas tanks should have defined oxygen concentrations balanced with nitrogen that a certified standard for oxygen content or for anoxic conditions. Pure nitrogen use a two stage regulator with the second stage set to the desired pressure. See the instructions in the written protocol for selecting an appropriate flow rate.
Then connect the tubing from the two stage regulator to the flow tube that controls the flow rate of gas entering the chamber. The outlets of the flow tube is connected to a gas wash bottle with fritted cylinder containing distilled water. Then connect the tubing on the other side of the wash bottle to one of the fittings on the environmental chamber.
This will hydrate the gas entering the environmental chamber. Use vacuum grease to seal the chamber. Then place weights on the lid of the chamber to ensure an airtight seal.
Test the seal by holding a small amount of water in the palm of a gloved hand to the gas exhaust fitting on the chamber and checking for bubbles. First, generate synchronized populations of sea Elgan. Place one to 100 GR adults in a drop of alkaline bleach solution on the surface of an unseeded nematode growth media plate.
Allow the bleach solution to absorb into the plate after an incubation period of at least eight hours. Transfer the synchronized L one larvae to plate seeded with live OP 50 bacteria. Alternatively, allow GRA adults to lay eggs on the plate for two to three hours to generate a group of worms that will develop synchronously or pick L four larvae from a mixed population to collect young embryos.
At the two to four cell stage, use a razor blade to chop GR adults in a small volume of water and then transfer the embryos by mouth pipette before transferring the worms to the plate for exposure to hypoxic conditions. Place a ring of 10 milligram per milliliter palmitic acid in ethanol around the edge of the plates. To prevent the worms from escaping the surface of the agar plate.
The palmitic acid will come outta solution. As the ethanol evaporates forming a physical barrier to ensure uniformity, be sure to replace the water in the gas wash bottle before exposure in the environmental chamber. After transferring the worms to the plates, seal the plates in the environmental chamber, initiate gas flow, and maintain exposure for the desired time to assay.
For the viability of embryos to adulthood, allow the worms to develop 48 hours after the return to room air, at which point they should be. Fourth stage larvae or one day adults score for viability to adulthood. To visualize worms pose hypoxic exposure, transfer the worms to a drop of M nine on a 22 millimeter square cover slip and invert onto a pad of 2%aros in M nine if necessary.
25 millimolar of amol or 10 millimolar sodium azide can be used as anesthetic. Alternatively, after exposure to hypoxia, the transparent environmental chamber can be placed directly on the stage of a dissecting scope. Two views are shown, one, including the entire gas flow setup.
The other with just the chamber on the microscope stage. Grow Bristol N two worms on four 10 centimeter high growth plates until the majority of the worms are GR adults. Wash the worms into a 15 milliliter conical tube with a one to five alkaline bleach solution and incubate with rotation until the worms begin to dissolve.
Not more than five minutes. Wash the worms three times with M nine spinning down at 1, 500 times G between each wash with no breaking plate. The bleached embryos onto 8 150 millimeter NGM plates and allow the embryos to develop into L four larvae for around 48 hours.
At 22 degrees Celsius, move the plates into the environmental chamber and expose to hypoxic and anoxic conditions for four hours or for the duration specified by the experimental design. To harvest samples quickly remove the lid to the hypoxic chamber. Take one sample plate and reseal the chamber time and record the steps.
After removing the worms from hypoxia, this entire process should take less than two minutes. Use dis distilled water to wash the worms onto a nylon filter and then pour into a labeled 15 milliliter conical tube containing 100 microliters of 1%SDS centrifuge. The worms in SDS in a desktop centrifuge are 1, 500 times G for 15 to 20 seconds.
With break, use a vacuum to remove most of the supinate from the tube, leaving the worm pellet untouched. Using a pipette transfer the worm pellet with a predetermined volume of liquid to a labeled 1.5 micro refuse tube. If the sample is to be used for SDS page, this tube can contain an equal volume of two times protein loading dye seal the tube and immerse in liquid nitrogen.
Repeat until all samples have been isolated. Follow these procedures for house air control samples for consistency samples can be stored at minus 20 degrees Celsius. This figure shows the viability of embryos exposed to 1000 parts per million oxygen, 5, 000 parts per million oxygen and normoxia around 210, 000 parts per million of oxygen.
The embryos are exposed to each condition for 24 hours in continuous flow oxygen chambers. Worms were moved to normoxic conditions allowed to develop to adulthood for 48 hours, and then scored for viability to adulthood. Following this procedure, samples can be collected for subsequent analysis by Western blot QPCR or physiological assays such as lifespan or progeny production.
After watching this video, you should have a good idea how to set up a continuous flow hypoxia chamber in your own lab.
