This research can help answer any questions in them biochem count and methodology field such as an analysis of development rights in nematode. The main advantage of this technique is protein precipitation in extraduction and drink and homogenization of the worms. Start this experiment by growing E.coli OP50 strain in 300 milliliters of LB broth liquid medium at 37 degrees Celsius overnight.
Store successful OP50 at four degrees Celsius. Pipette two milliliters of the cultured OP50 on minimum three previously prepared NGM, or Nematode Growth Media Agar Plates and spread by hand swirling the plate. Incubate the plates at room temperature overnight to create a thin layer of OP50.
Next, use a sterile toothpick to pick a piece of agar from a plate with worms, to add at least 100 worms to each OP50 NGM plate. Culture at least three plates with the worms at 20 degrees Celsius, until the worms reach adult stage, which is confirmed under stereoscopic microscope. Locate gravid hermaphrodites filled with eggs using a stereoscopic microscope.
After adding five milliliters of S-buffer to plates, use Pasteur pipette to transfer the worms from all three plates to a 15 milliliters conical tube. Wash the worms three times by adding 15 milliliters of S-buffer and then centrifuging at 300 times G at room temperature for 30 seconds. If the volume of S-buffer exceeds five milliliters, remove the excess buffer after centrifuging.
For successful exonization and bulk egg isolation, dissolve the worms in 0.5 milliliters of alkaline hypo chlorate solution. Mix by inverting and incubate at room temperature for 10 to 15 minutes. Centrifuge at 1, 400 RPM for 30 seconds to remove the solution.
Use 15 milliliters of S-buffer to wash the egg pellet. After centrifuging it remove the supernatant and repeat this wash at least three times. After the last wash, we suspend the pellet in five to six milliliters of S-buffer, without E.coli and incubate the eggs overnight at 20 degrees Celsius to hatch them and achieve H synchronous culture of L1 stage larvae.
Pipette 10 microliters of S-buffer containing L1 worms on three cover slips. Count the larvae under a stereoscopic microscope and calculate the average to determine the approximate number of L1 stage larvae. Finally, transfer the L1 stage larvae to five NGM Agar Plates with OP50 and grow them at 20 degrees Celsius until the young adult stage, during which self-fertilization occurs and a few eggs are laid.
Observe the worms every day under the microscope and then collect the young adult, or five day old worms. To select only living worms, add the worms suspended in three to four milliliters of S-buffer to a 15 milliliters conical tube. Then add an equal volume of ice-cold 60%sucrose and mix.
Centrifuge the tube at 1, 500 times G at four degrees Celsius for 15 seconds. Then use a Pasteur pipette to carefully move the worms off the wall of the tube, and then remove the floating worms into a fresh tube. Fill the tube with S-buffer to wash the worms, and centrifuge at 1500 times G at four degrees Celsius for 30 seconds.
Remove the buffer and repeat the wash two more times. After the third wash, carefully remove the supernatant, making sure not to remove any worms, and use a 1, 000 microliter pipette tip to measure the wet volume of the washed worms. For protein precipitation, use a Pasteur pipette to add the washed worms to an equal volume of ice-cold 10%trichloroacetic acid in a homogenizer placed on ice.
Homogenize using 40 strokes of passel with rotation, up to 1, 300 RPM. Use an ultrasonic homogenizer with 20%duty cycle to sonicate the homogenate. Use a Pasteur pipette to transfer the homogenate to 1.5 milliliter tube placed on ice.
After transferring the homogenate to a micro centrifuge tube, centrifuge at 8, 000 time G at four degrees Celsius for 10 minutes to clarify it. Transfer the supernatant to a fresh tube and add four molar potassium chloride and incubate on ice for 20 minutes to neutralize it. Then centrifuge it at 8, 000 times G at four degrees Celsius for 10 minutes.
Transfer the supernatant to a fresh tube and store at 80 degrees Celsius until needed for further assays. To measure the concentration of lactate, use a colorimetric assay kit to carry out duplicate analyses for the test samples. Add 10 microliters of the test samples to each well of a 96-well plate, and then add lactate assay buffer to each sample to adjust the volume to 15 microliters.
Dilute 100 millimolar L+Lactate Standard with lactate assay buffer to one millimolar. Then, into a series of wells, add zero, two, four, six, eight, and 10 microliters of diluted L+Lactate Standard. Add 50 microliters of reaction mix to each well, and incubate at room temperature protected from light for at least 30 minutes for the color to develop.
Finally, use a microplate reader to measure the absorbance of each well at 570 nanometers. Subtract the absorbance of the background control mix from the absorbance of the reaction mix. Then plot the lactate standard curve, and calculate the lactate concentrations from the curve by multiplying each concentration by 2.25.
Use a colorimetric assay kit to measure the concentration of pyruvate in the test samples, and carry out duplex examinations. Add 10 microliters of the test samples to different wells of a 96-well plate, and add 90 microliters of working reagent to each sample. Incubate covered for 30 minutes at room temperature.
Then place the plate in the microplate reader to measure the absorbency of each well at 570 nanometers. Plot the pyruvate standard curve, and calculate the pyruvate concentrations from the standard curve by multiplying each concentration by 2.25. And finally, to measure the protein concentration in the test samples, use a colorimetric assay kit as described in the text protocol.
After protein precipitation, colorimetric assays can be used for the quantitative determination of lactate and pyruvate concentrations. This experiment shows the accuracy of colorimetric assays contrary to previous reports in C.Elegans. Furthermore, these assays are sufficiently sensitive to measure lactate and pyruvate concentrations even in small scale samples in a short period of time.
To accurately detect lactate and pyruvate in C.elegans, it is crucial to perform protein precipitation during homogenization of the worms. The amount of lactate and pyruvate detected is higher when samples are protein precipitated during homogenization, compared to intact cytosolic fraction or after homogenization when there was no lactate or pyruvate detected at all.
