Over the past decades, human lifespan is extended, due to improvements in standards of living and advances in medical science. Age is the biggest risk factor for many life-threatening diseases, including neurodegenerative disorders, such as Alzheimer's, Parkinson's and Huntington's disease. Here, we demonstrate methodologies utilizing some versatile new method models, including hyper-active ion channel induced necrosis and protein aggregates induces neurotoxicity, to monitor and dissect the cellular molecular mechanism of neural degeneration.
Peak healthful larvae of make four and make three Newton nematodes onto nematode growth media plate, sieving with E.Coli using dissecting stereo microscope. Place ten and four nematodes, parasitic enzyme plate and grow them at the standard temperature, of 20 degree celsius. Five days later, wash the plate with 1 mL and 9 buffer and collect the animals in 1.5mL tube.
Centrifuge at 30, 000 g for 30 seconds and remove the supernatant. Add 0.5 mL of plating solution. Plating solution is stored at the room temperature.
Vortex and monitor periodically until they all have dissolved. Avoid plating for periods longer than five minutes. Centrifuge at 10, 000 g for 30 seconds and remove the supernatant.
Wash twice the palette with 1 mL and nine buffers. Centrifuge at 10000 g for 30 seconds and remove the supernatant. After washing, resuspend the eggs in 200 microliters M9 buffer and incubate them for 25 minutes, at 34 degrees Celsius in a water bath.
Maintain a separate group of eggs at 20 degree Celsius. Pipette 100 microliters containing control or heat shock treated eggs and place them on unseeded 10 GN plate. Each plate contains at least 100 to 200 eggs.
Incubate the eggs at 20 degree celsius until hatching. Use and M9 buffer to wash plates and collect L1 life and nematodes in 1.5 ml tube. Centrifuge at 10, 000 g for 30 seconds.
Remove the supernatant and keep the palette. Add 100 microliters of 20 millimolar M9 levangial buffer to anesthetize the nematodes. Pipette 10 microliters of M9 levangial buffer, containing L1 watch and mount them in 2%agar-ls spot place gently a coverslip on the top of the sample.
Observe watch using the DIC microscopy. Scanning degeneration of the 630 receptor neurons by counting cells with a characteristic virtual aided appearance of that nematode. Synchronize nematodes by selecting and transferring 15 to 20 L4 larvae of each in strain on firstly bacteria C then GM plates.
Incubate and let the nematodes to grow at the standard temperature of 20 degrees Celsius. Perform daily preconditioning for 30 minutes by transferring the plates in an incubator set at 34 degrees Celsius. Then return the preconditioned nematodes back at the standard temperature of 20 degrees Celsius.
Add 10 microliters of 20 millimolar M9 levangial buffer drop at the center of the agar-ls spot. Pick the respective transgenic nematodes and transfer them in M9 levangial drop. Place 20 to 30 nematodes the per drop.
Place gently cover slip on the top of the sample. Seal the cover slip with nail polish to preserve humidity throughout the imaging process. Using a fluorescence microscope combined with a camera, detect and capture your study images of the head region at 20 x magnification.
Monitor seven days of transgenic nematodes for dopaminergic neuronal cell death. Measure the neuronal policul aggregates in the head region of four days old transgenic animals expressing 240 fusion with live people. Necrotic cell death induced a hyperactive ion channels is the mainest in nematodes that has former heat shock precondition eggs.
Heat shock preconditioning, promoting a prediction against alpha-synuclein induced cell death in seven-day old adult hermaphrodite and decreases Q40 protein aggregates in the head region of four day old adults. Aging is a consequence of damage caused by gradual degeneration of cellular and tissue homeostasis. Thus understanding of manipulating age-related neuronal break down are top places priorities.
The presented techniques combined with genetics and pharmacological screens could lead to a notification of normal cell death modulators with potential therapeutic interest.
