DNA Damage Test

''This procedure is used to evaluate the sensitivity of Tetrahymena cells to DNA damaging agents. ''

''Use extreme caution when working with DNA damaging drugs because most are known carcinogens. Wear masks and gloves at all times.''

Materials

 * Tetrahymena cultures (fresh)
 * DNA damaging drug (e.g. cisplatin or MMS)
 * SPP or NEFF medium
 * Starvation Buffer ( 10 mM Tris-Cl, pH 7.5)
 * Tube-needle apparatus

Treating Cells with DNA damaging drugs

 * 1) Place 1 mL of cells in each well of a 24-well plate.
 * 2) Add DNA damaging drug to the appropriate concentration to each well.
 * 3) Gently swirl the 24-well plate to mix the drug in the media and incubate the plate at 30 °C for 4 hrs.
 * 4) During the 4-hour incubation time make the appropriate number of drop plates using SPP or NEFF media. These will be used after cells have been treated and washed.

Assaying DNA Damage in Vegetative Cells

 * 1) Place the cells for each well into 1.7 mL tubes.
 * 2) Spin at 1,100×g for 2 minutes at room temperature in microcentrifuge.
 * 3) Pour off supernatant quickly as cells will start swimming up into the media.
 * 4) Add 1 mL of Starvation Buffer and gently resuspend cells (use your finger to flick the bottom of the tube).
 * 5) Repeat washes 2 more times.
 * 6) After the final spin resuspend cells in 0.25-0.5 mL of Starvation Buffer.
 * 7) Take a previously made drop plate, and place one cell in each drop using a Needle Bulb apparatus (do not use the Needle Tube). Mark any unused drops or drops known to have multiple cells on the plate – these should be eliminated from further analysis.
 * 8) Incubate for 24 hours at 30 C.
 * 9) Count the number of live cells in each drop. Also count number of drops with live cells vs. drops with dead cells.  Record data.