In the C.elegans field, this method can help answer key questions which require the production of large nematode populations and samples using plate culture. The main advantages of this protocol are its easy establishment, and its ease to description, including pitfalls and applications. After obtaining a synchronized population of nematodes, add approximately one milliliter of M nine buffer to the plates, and carefully distribute the buffer by swinging the plates.
After this, collect the nematodes in a 15 milliliter tube. Now, invert the tube and use a microscope to evaluate the density of the nematodes. Adjust the density of the suspension as needed, and continue to evaluate it under the microscope.
Apply 150 microliters of the suspension directly to the plates with modified pipet tips. To ensure an equal distribution of nematodes between the plates, invert the tube of suspension at regular intervals. First, wash the nematodes using a modified pipet tip with approximately 500 microliters of M nine buffer.
Using the same buffer, continue detaching the nematodes until most of the animals are collected in suspension. Next, take up the nematodes with a modified pipet tip, and transfer them to a plate with OP50. The modified pipet tips decrease shear stress and damage to the nematodes because of a larger diameter.
Then, allow the plate to dry. Before collecting samples, prepare liquid nitrogen to snap freeze the collected nematodes. Then, place M nine buffer on ice.
Apply one milliliter of M nine buffer to each plate, and gently swirl to wash each plate. Then, take up the volume of M nine buffer, and transfer it to new 15 milliliter tubes. Centrifuge the tube briefly at 800 times g, before removing most of the M nine buffer.
Next, wash the samples three times with 10 milliliters of M nine buffer to remove any bacteria. Using a glass pipette, transfer each pellet to an empty 1.5 milliliter tube. Use the glass pipette to take up one milliliter of M nine buffer from the second 1.5 milliliter tube, to rinse the nematodes from the pipette wall.
Then, wash the remaining nematodes from the 15 milliliter tubes, and transfer them to the sample tubes. Next, centrifuge the sample tubes at 19, 000 times g for one minute, and remove as much supernatant as possible. Then, seal each tube with paraffin film, and snap freeze them in liquid nitrogen immediately.
Store the tubes at minus 80 degrees Celsius until lysate preparation. Add the same volumes of 0.5 millimeter zirconium oxide beads, and twice the volume of lysate buffer with proteinase inhibitor cocktail to each frozen sample. Then, place the tubes in a homogenizer for one minute on speed nine.
Centrifuge the samples for 30 minutes at four degrees Celsius, and 19, 000 times g. Finally, transfer the supernatant to a new tube on ice, and store it at minus 80 degrees Celsius for future experiments. In this protocol, large quantities of C.elegans were cultured for applications in diabetes research.
The number of nematodes collected correlated to the protein yield of the lysate, demonstrating that reproducible quantitative protein isolation can be achieved using this protocol. Mass spectrometric analyses of the reactive metabolite methylglyoxal, and advanced glycation end products formed by methylglyoxal exposure are shown here. Glucose-treated nematodes and controls were compared.
Transferring nematodes via pipette can create shear stress, potentially harming the nematodes. Oxidative stress levels in pipetted nematodes and nematodes transferred with a metal wire did not differ using this protocol. Following this procedure, other methods like western blot can be performed, in order to answer additional questions in proteomics.
This protocol also facilitates experiments that relate multiple parameters to the same population.
