20.109(S18):Induce DNA damage and apply drug treatments for cell viability and identification of regulatory motifs in RNA-seq data (Day8)

Protocols

Part 1: Induce DNA damage and apply treatments

You will assess cell viability in response to DNA damage and drug treatment to investigate the conditions used when the RNA-seq data was generated. In this exercise, you will induce DNA damage in the cells that were seeded by the teaching faculty then add the drug that you selected on M2D2. Next class you will examine survival of your cells using an assay the abundance of live cells.

1. Prepare your working space within the tissue culture hood.
2. Calculate the volume of etoposide stock needed for DNA damage induction.
   • Obtain an aliquot of pre-warmed media from the 37 °C water bath (11 mL).
   • Determine the volume of etoposide stock (100 mM) you need to add to the media for a final concentration of 100 μM.
3. Retrieve your 12-well plates from the 37 °C incubator and visually inspect your cells with a microscope.
   • Record your observations concerning media color, confluency, etc. in your laboratory notebook.
4. Move your plate into the tissue culture hood.
5. Aspirate the spent media from each well.
   • Be careful not to cross-contaminant between wells with different cell lines!
6. Add ~1 mL of PBS to each well and rock the plate gently to wash the cells.
7. Aspirate the PBS from each well.
   • Again, be careful not to cross-contaminate.
8. Add 1 mL of PBS to well A1 and A3.
   • These wells will be the 'no DNA damage' controls for the DLD-1 and BRCA2- cells.
9. Add 1 mL of the media containing etoposide that you prepared in Step #2 to the empty wells.
10. Carefully put your plate in the 37 °C incubator for 60 min.
11. Assist you partner if they are still working. When you are both done, return to the main laboratory space.
12. Return to the tissue culture space and retrieve your plate from the 37 °C incubator.
13. Aspirate the PBS or media containing etoposide from each well.
    • Be mindful of cross-contamination.
14. Add 2 mL of fresh media to each well.
15. Calculate the volume of drug you need to add to each well for the final concentrations shown on the figure below.
    • Be sure to use the concentrations appropriate for the drug you chose.
    • The stock concentrations of mibfradil and loperamide are 10 mM.
    • It may be helpful to dilute the drug stocks to avoid pipetting very small volumes.
16. Add the appropriate volume of drug and move your 12-well plate to the 37 °C incubator.
17. Assist your laboratory partner, if necessary, then clean the hood and return the main laboratory space.