Timing of Cycles

• 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.

6. Aspirate all but 0.5 ml of the PBS from the tube. Gently stir the cells by tapping the centrifuge tube and add 5.0 ml of freshly prepared fixative, drop by drop, gently mixing between each drop. Allow the cells to fix for 1-2 hours.

7. Collect the cells, rinse once with fresh fixative and pellet cells into a final volume of about 0.5 ml of fixative.

8. Use a pasteur pipette to transfer the cells onto clean, subbed slides and allow to air dry.

9. Prepare slides for autoradiographic analysis as in Exercise 11.4. Coat with nuclear track emulsion and expose for one week. Develop autoradiograms and stain lightly with Giemsa as directed in Exercise 11.4. Prepare permanent slides by attaching coverslips with Permount.

10. Examine the slide and count the total number of cells in interphase and the number of cells that are radioactively labeled. Express this number as a decimal fraction (i.e. if 45% of the cells are labeled, the fraction is 0.45).

11. Use the following formula to compute the length of the S phase.

Time for S phase = Mean Cycle Time x Fraction of Labeled Cells

Optional

The entire process can be repeated with exposure to the radioactive thymidine followed by a brief period of exposure to non-radioactive thymidine. Fix a series of cultures at half hour intervals after removal of the pulsed radioactive label. Expose the cultures to nuclear track emulsion and examine for labeled mitotic images (as opposed to interphase cells). The time between the appearance of the first mitotic cells with label and the level of 50% of the mitotic images labeled represents an approximation of G2.

The length of the mitotic division can be measured directly with phase contrast microscopy (usually less than 1 hour). Estimate G1 by subtracting the time for mitotic division, G2 and S from the Mean Cycle Time.