High-throughput Phenotyping Assay Andersen laboratory - updated August 2018 BCR Amplifying your populations: - For each generation picked, use plates from the same batch of NGMA and OP50. - If picking for a GWA assay, make sure there will be enough 6 cm plates for each generation. - Pull out plates to room temperature several hours (or the night) before picking to ensure you will not be picking to “wet” plates. - It is necessary monitor your strains through this whole process. If they are growing slowly you can leave the worms at room temperature the day/night before you pick them. Start monitoring at the first generation to make subsequent picking as easy as possible. Be careful of temperature sensitive strains or mutants, as they are most affected by shifting temperatures. - These steps ensure that trans-generational effects of starvation and high levels of crowding are minimized. 1. Using sterile, autoclaved spatulas, chunk a starved plate onto a fresh OP50-spotted plate. This step is usually done on Wednesday/Thursday for normal sorter assays (see schedule at the end of this protocol). If the plates you are chunking from are several months old, you should chunk a larger area of agar to the new plate to ensure that enough worms come off of the chunk. Store your plates in the 20˚ room between generations, unless worms are growing slowly, in which case store your worms at room temperature on your bench. 2. After 48 hours, pipette 15 μL bleach solution away from the lawn of a new plate. Pick ten to fifteen gravid worms into the bleach. Do this for each strain. This will help prevent the spread of contamination from the chunk plate. 3. After 24 hours, pick 10-15 L1s to a new plate for each strain. If for some reason the L1s didn’t survive the bleach, make a note of it and pick from the chunk plate for generation one. 4. After 48 hours, pick five late-stage L4s to one 6 cm plate from each strain. This pick is generation one. NOTE: Worms may have grown into young adults by this time. Pick the youngest worms you can find. 5. Three days later, pick five late-stage L4s to a fresh plate for each strain. This pick is generation two. 6. Three days later, pick five late-stage L4s to fresh plates for each strain. Make sure all of the plates are from the same batch of NGMA and same OP50. This pick is generation three. • Dose responses pick to four plates. • Heritability/other high replication assays (NILs, allele swaps) pick to two or three plates. • Mappings pick to one plate. 7. Four days later, pick five L4s of each strain to two plates for every well that will be setup in the high- throughput assay. This is generation four. Pay attention to the timing for each strain. Some strains grow more slowly. It is best to group strains together with similar growth speeds. We’ve found that it is often good practice to note the time and date that the plate is setup. In that way, you can see how long it takes to get to a plate of gravid worms without starvation. If strains are growing slowly or have a low population number, pick six or seven worms per strain. Try to pick as fast as possible. You don’t want one strain to be four hours older than the last strain. • Dose responses - you will have five wells for each of four strains in quadruplicate. Therefore, you will have 20 wells and require a total of 40 plates from each strain. Write down the order that you picked each strain from one to 40. In that way, you can mix older plates with newer plates to even out the age of the worms picked. If you will be running less than ten 96 well plates in your assay, you can reduce the number to 14-20 plates/strain. • Heritability assays - you will have seven wells for each of 12 strains. Therefore, you will need 14 plates from every strain. • Mappings - you will have one well for each strain. Therefore, you will only need to pick two plates of every strain. 96-well Bleaching and Synchronization: 8. In the afternoon, four days before bleaching (~84-90 hours, the same day as your gen. 4 pick), make 200mL of fresh bleach solution (see recipes page). Store at 4°C until needed. 9. Make 500 mL batches of fresh K Medium (see recipes page) a few days in advance of preparing assay plates so you can check it for contamination. This can be made the same day as the bleach. When making K medium, add all water and salts, then filter with Thermo Filter Unit (Cat #566-0020). After filtering, add cholesterol that has been filtered (Cat. # PN 4192, Pall Life Sciences). Decrease any opportunities for the filtered K medium to become contaminated. If your K medium is contaminated, make new. 10. If you are doing a 96 well bleach, check that there are enough autoclaved deep 96 well plates and autoclaved pasteur pipettes before your bleach day. They must be cleaned at least a day in advance so they have time to dry before autoclaving. Check deep 96 well plates for any white residue at the bottom of the wells, as these may contaminate your assay. 11. Pay close attention to your strains as they grow during this last generation. On the day of the bleach, you want to have a healthy robust population of gravid animals. Check the plates two and three days after picking to make sure that they will be staged for the bleach and staged close to each other. Usually, we take out a few plates (or the entire batch) for 12 to 24 hours to speed up slow strains. 12. Four days after picking generation four, check the strains to ensure your worms are staged properly for your bleach. We want a plate with many gravid animals but not a huge population size and no starvation. 13. Type up how you would like your strains to be organized in each 96 well plate (see plate setup examples at the end of this protocol). You will later use this information to make a CSV file used in data analysis. Once your worms are staged properly, sterilize your bench with bleach and ethanol, then lay out your fourth generation plates in the same order you will have them arranged in your 96 well plates. NOTE: if your assay has 12 or fewer strains, you may choose to perform a conical bleach instead of the 96 well bleach. Follow the separate protocol attached. 14. Take your bleach solution out of the 4º fridge before you start washing your plates into the deep 96 well plate (or 30-45 minutes before you will finish filling the wells of the deep-well plate). This allows the bleach to warm up a little bit, allowing for more effective bleaching. 15. Working with a partner, use M9 to wash worms off from plates and add to the appropriate well. Add two mLs of M9 to the first plate using a filler dispenser pipette. Gently shake the M9 back and forth in the plate, dump the M9 onto the next plate and shake the M9 back and forth. Continue this for each plate needed for one well (normally two plates per well). After the final plate, have your partner transfer the worms using a Pasteur pipette to the corresponding well of a deep 96-well plate (Arctic White, AWLS-X10005). You can use a piece of foil wrapped around the deep-well plate to help keep your place. It is important to make sure this step goes efficiently and quickly. The longer the adults and embryos stay in the hypoxic condition, the more a stress response might be triggered. • It is necessary to add the same volume to each well so that the 96-well aspirator functions properly. This is why you should only add M9 to a set of plates directly before you wash the worms from those plates into a well. If liquid remains on the plates for too long, it will absorb into the plate, giving different final volumes. 16. Carefully remove the Pasteur pipette bulb and leave the pipette into the well to mark the well that has been filled. 17. After washing all worm strains into the deep-well plate, remove all Pasteur pipettes. Be careful to avoid cross contamination, by slowly removing them one at a time and allowing excess liquid to drain into the well. You can again use aluminum foil to help keep wells covered once the pipettes are removed. Put the pipettes into 50% bleach solution until the setup is complete. 18. Make sure all wells, including the blanks have an equal volume of liquid. It is easier to fill them and make even now, then when you are adding bleach to mostly empty wells. This step is crucial to allow the 96-well aspirator to remove liquid from each well evenly. Add ~1.4 mL to wash wells with the dH2O squirt bottle to ensure that they are not problematic during aspiration. Securely place the sealing mat on the deep-well plate. Check to make sure the sealing mat is flat across the whole plate. 19. Set up the 96-well aspirator following the Setup 96-well Aspirator protocol (i.e. clean the aspirator with ethanol if required). Empty the vacuum flask attached to the 96-well aspirator. It works more reliably and reproducibly when empty.