Chapter 11: Cell Cycles
Exercise 11.4 - H-Thymidine Uptake by Cultured Cells
Fibroblast cells in log phase growth
Ca, Mg free-phosphate buffered saline (PBSA)
5% (w/v) Glutaraldehyde (GTA)
2% (w/v) Perchloric Acid (PCA)
Subbed slides (coated with chrom alum gelatin) and Permount
Nuclear Track Emulsion (Kodak or Ilford)
Darkroom and chemicals for photographic processing
Giemsa stain, graded series of alcohols, xylol
- Dektol developer
- Kodak Fixer
- Grow either L cells (Mouse fibroblasts) or chick embryo
fibroblasts on coverglasses and then give them
for a short period of time.
- At the end of the labeling period, wash the coverslips in
PBSA by gently grasping a coverslip with forceps and passing it
through a beaker of saline.
- Fix cultures in glutaraldehyde for 15 minutes.
- Wash in several changes of water.
- Wash in cold 2% PCA for 5 minutes to remove unincorporated
labeled precursors to DNA. Repeat twice.
- Wash in water 5 minutes. Repeat.
- Dry the backs of the coverslips with filter paper, and mount
CELL SIDE UP on slides with Permount.
The slides should be very clean. Coat the slides beforehand with
chrom alum gelatin (CAG) by dipping the slides into CAG solution
and draining until dry. This coating, and the gelatin coating on
the coverslips, help to prevent the emulsion layer (below) from
pulling away from the slide during later development.
- Allow Permount to dry overnight.
- In a darkroom, spoon out a small amount of gel into a
suitable vessel, and slowly melt it at 45° C in a water bath.
Kodak NTB-3 emulsion is stored refrigerated as a gel in a screw
cap bottle inside a double light-tight box.
- Dip the slide in the emulsion and drain momentarily. Place
the slides vertically on a test tube rack in an oven set at
28° C for at least 1 hour in darkness.
In general, the slides should be dried at a temperature greater
than will be used for developing. This minimizes undesirable
separation of emulsion from the slide.
- Place the slides in light-tight boxes containing dessicant
and store at 4° C.
- Develop a sample slide in Dektol diluted 1 part developer to
2 parts distilled water at 18° C for 90 seconds. The
temperature of the developer will control the size of the silver
grains. Increased length increases background fog of development.
Develop the slides as indicated in Chapter Two. The length of
exposure (refrigerator storage) must be determined for each
system (a function of specific activity of label in medium, pool
sizes, length of labeling, synthetic rate, etc.). Thus extra
control slides are always included to allow repeated sample
developing until a useful number of silver grains have
- Pass the slides through two changes of distilled water, and
into photographic fixer at 18° C. Fix for 5 minutes.
- Wash slides in two changes of distilled water, for a total
of 5 minutes.
- Stain the cells with Giemsa diluted 1:30 as required, or dry
slides slowly in a dust-free atmosphere, and stain later.
- Wash off excess stain briefly in distilled water, dip slide
briefly in 70% ethanol, dehydrate in 95% and 100% alcohol, and
clear in xylene. Mount coverslip with Permount.
- Examine the slides with a microscope at 10X magnification
and look for clusters of silver grains over the cells. Count and
calculate the percent of cells that are labeled.
- Examine the slides at 40X magnification. Count the number of
grains per cell.
- Prepare a histogram by plotting the number of cells versus
the number of silver grains.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- email@example.com