20.109(F20):M1D2

Part 3: Seed cells for gamma-H2AX assay

1. Obtain one 12-well plates from the front laboratory bench.
   • Clearly label the plate with the date and your team information. Include the name of the cell line seeded!
2. Carefully place a coverslip into each of the wells EXCEPT C1 and C2 (the bottom row, column 1 and 2).
3. Add 2 mL of fresh media to each well that contains a coverslip.
4. Calculate the volume of cells that contains 25,000 cells for each cell suspension (25,000 cells / well).
   • Use the density you calculated above to determine the volume.
5. Mix the cells and add the appropriate volume of cells into each of the wells.
   • CHO cells settle quickly in conical tubes. It's important you mix before adding cells to the 12 well plate.
6. To ensure that the cells are evenly dispersed in the well, slide the plate up / down and left / right 3-5 times.
7. Carefully move your plates to the 37°C incubator
8. Clean out the tissue culture hood:
   • Aspirate any remaining cell suspensions.
   • Dispose of all vessels that held cells in the biohazard waste box and be sure that all sharps are in the sharps jar.
   • Remove any equipment or supplies that you transferred into the hood and return to the appropriate location.
     • Please leave the equipment that was already there.
   • Spray the TC hood surface with 70% ethanol and wipe with paper towels.
     • Be sure the paper towels are disposed of in the biohazard waste box!
   • Empty the benchtop biohazard bucket into the biohazard waste box.