Timing of Cycles

Materials

Monolayer cultures grown in 75 mm culture flasks (Cells from Exercise 11.4 may be used, or cultures of tetrahymena, yeast, or algae may be used.)

H-thymidine with at least 4 µc/ml, 0.36 c/mM

Phosphate buffered saline (PBS), Trypsin

Methanol:Acetic acid (3:1) fixative

Nuclear track emulsion and equipment for autoradiographic analysis

Subbed slides, coverslips, permount

Giemsa stain

Microscope (Phase contrast if total cycle is to be measured)

Clinical centrifuge

Procedure 3

1.Expose log cultures of cells 4 to H-thymidine for a period of 30 minutes. The Mean Cycle Time (hours) of the culture must be known. 5 Calculate the MCT by plotting the growth of the culture and determining the average time for the cell population to double.

2.Pour off the radioactive media (discard with radioactive wastes) and wash cells twice with PBS. Add washings to radioactive waste.

3.Add 1.5 ml of 0.25% trypsin to the flask to dislodge the cells from the flask. Add 10 ml of PBS, mix and pour into a centrifuge tube.

4.Centrifuge in a clinical centrifuge at 600 RPM for 5 minutes to pellet the cells.

5.Aspirate the supernatant, leaving about 0.5 ml of cells packed in the bottom of the tube. Resuspend in PBS to wash, and collect again by centrifugation at 600 RPM for 5 minutes.