Exercise 11.5 - Timing of Cycles
Figure 11.8 Timing of mitotic cycle
- 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
- Subbed slides, coverslips, permount
- Giemsa stain
- Microscope (Phase contrast if total cycle is to be measured)
- Clinical centrifuge
- Expose log cultures of cells 4
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
- Pour off the radioactive media (discard with radioactive
wastes) and wash cells twice with PBS. Add washings to
- 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 in a clinical centrifuge at 600 RPM for 5 minutes
to pellet the cells.
- 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.
- 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.
- Collect the cells, rinse once with fresh fixative and pellet
cells into a final volume of about 0.5 ml of fixative.
- Use a pasteur pipette to transfer the cells onto clean,
subbed slides and allow to air dry.
- 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.
- 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).
- Use the following formula to compute the length of the S
Time for S phase = Mean Cycle Time x Fraction of Labeled Cells
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.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org