Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

The overall goal of this procedure is to measure oxidative stress, resistance of ceno Titis elegance, nematodes in liquid. This is accomplished by first dissolving ROS inducing agents such as paraquat in M nine Buffer and Ali quoting the solution and a 96 well microtiter plate. The second step is to transfer five to eight synchronized late L four nematodes to every well using at least 12 wells per condition.

Next, using a dissecting microscope, the number of living and dead animals is determined every hour until most animals are dead. The final step is to calculate the percentage of animals that survived at every time point for every condition at least six days prior to performing the oxidative stress resistance assay. Prepare the modified young grins only bact PEPIN or MYOB dry mix for making the MYOB medium mix well with shaking.

To prepare one liter of MYOB medium, combine six grams of the MYOB dry mix with 17 grams of agar and make up to one liter with H2O autoclave for 45 minutes at 122 degrees Celsius. Pour the MYOB medium into plates and let the plates dry at room temperature for two days using sterile techniques. Inoculate 100 milliliters of LB broth medium with e coli, OP 50 bacteria and grow overnight at 37 degrees Celsius on the following day.

Seed MYOB plates with the E coli OP 50 bacteria allow the bacteria to grow at room temperature for 48 hours. In cases where gene downregulation will be accomplished by RNA interference, start the procedure by preparing M-Y-O-B-R-N-A-I feeding plates. After autoclaving, the MYOB medium allow it to cool down to about 55 degrees Celsius.

Then add isopropyl beta D one thito peroxide, or IPTG and ampicillin to the MYOB medium. Pour the plates and let them dry at room temperature for two days streak E coli HT one 15. Bacteria on agar plates containing ampicillin and tetracycline.

One strain has been transformed with a plasmid that expresses control empty vector or ev. Another strain has been transformed with a plasmid that expresses the double stranded RNA that targets the FOLLIN one. Mr.NA grow overnight at 37 degrees Celsius on the following day.

Pick colonies and inoculate LB medium containing ampicillin. Grow the cultures overnight at 37 degrees Celsius. See the MYOB RNAi plates with e coli HT one 15 EV bacteria or HT one 15 Collin one RNAi bacteria keep the plates at room temperature for 48 hours before usage.

Prepare several plates based on the number of worms needed during the assay, and allow the worms to lay eggs for six hours at 20 degrees Celsius. Next, use a platinum wire worm pick to remove the mothers. Check the plates under the microscope to assess the number of eggs laid.

Allow the eggs to hatch and the worms to grow at 20 degrees Celsius for 48 hours. 48 hours later, the animals should be in the late L four or young adult stage. Note that the 48 hour incubation only applies to mutants that grows similarly to wild type animals.

If a new mutant is to be tested, it is important to first monitor its developmental rate. In this demonstration, oxidative stress will be induced using paraquat. Prepare a fresh 100 millimolar paraquat solution as described in the accompanying protocol text.

Paraquat is a hazardous and highly toxic compound and must be handled according to appropriate guidelines. Pipette 40 microliters of the paraquat solution to every well of a 96. Well plate use at least 12 wells as replicates of every condition tested using a platinum wire worm pick.

Transfer five to eight L four larvae animals to every well indicate the start time and the end time for every condition tested place the 96 well plate at 20 degrees Celsius an hour after the start time score dead and alive animals using a dissecting microscope gently shake the plate before starting to count. Worms that do not move even after spotting a high intensity light are scored as dead. Look at worm shape tails and head movement at high magnification to distinguish dead worms from live worms.

Score survival every hour until most worms are dead, and repeat this assay at least three independent times to determine the survival percentage for each time point. First, calculate the total number of animals per condition by adding the total number of animals transfer to each. Well ignore the animals that were damaged or killed upon.

Transfer next for every time point. Calculate the total number of dead animals per condition. Calculate the percentage of death and the percentage of survival in this study.

100 millimolar. Paraquat was used to determine the resistance of wild type C elegance compared to a follin one mutant that has been shown to resist oxidative stress, heat, and anoxia. After four hours of treatment, 48.3%of wild type survived as compared to a 77.8%survival in mutant animals.

Similar results were obtained in a comparison between wild type animals fed with control, empty vector or follin. One RNAi bacteria downregulation of follin one using RNAi increased the resistance to 100 millimolar paraquat demonstrating that downregulation of gene function using RNAi mimics loss of function mutations Once mastered, this technique can be easily performed to determine genes that alter resistance to oxidative stress and see elegance. And finally performing these screenings and see elegance has a great value and potential in the treatment of human diseases linked to oxidative stress.