HARVESTING OF FIBROBLAST CELL CULTURES
Contributed by San Diego Zoo's Institute for Conservation Research, Genetics Division
Materials and Reagents Needed
 6%CO2 incubator set at 37° C
 37° C H2O bath
• actinomycin D (AMD): working solution 100 µg/ml
• Velban: working solution 0.5 µg/ml
• ethidium bromide stock solution 10 mg/ml
• colcemid: working solution 5.0 µg/ml
• 15cc centrifuge tubes
• Hank's balanced salt solution (HBSS)
• Trypsin-EDTA solution
• 0.075 M KCl 5.59 g KCl/1 liter dH2O, sterile filter
• Carnoy’s Fixative: 1 part glacial acetic acid: 3 parts absolute methanol
• ice bath (optional)
• siliconized Pasteur pipets (5-3/4") and bulbs
Protocol
1. Check secondary cultures for optimal growth of cells. This usually occurs
approximately 24 hours after a 1:2 passage. Evidence of mitotic doublets and
rounded-up cells should be evident and abundant.
2. To each T75 flask add 100 µl colcemid for 15 min- 1 hr.
3. Transfer culture fluid to a 15ml centrifuge tube. Add 1-2 ml HBSS to the flask to
rinse all medium out. Pour this rinse into your centrifuge tube. Add 1-2 ml
Trypsin-EDTA to the flask and make sure all cells get coated. Pour Trypsin-EDTA
into centrifuge tube, leaving a tiny amount still in flask. Wait a few minutes. Rap
sides of flask gently to detach cells. Check under microscope to make sure all cells
are removed. Add 3 ml HBSS and rinse all cells out.
4. Centrifuge 10 min at 1000 -1200 RPM. Remove supernatant, leaving
approximately 0.5 cc. Resuspend pellet gently.
5. Add 7 ml 0.075 M KCl warmed to 37° C. Resuspend gently. Incubate in 37° C H2O
bath for 10-30 min. Incubation time varies for different cultures; you will have to
find the best times by trial and error. Meanwhile, make fixative. Place on ice bath
(optional).
6. Add 3 drops of cold fix to the hypotonic solution while gently agitating the tube.
Resuspend the solution.
7. Centrifuge at 1000 RPM for 10 min.
8. Remove all but 1 cc supernatant. Resuspend well. Carefully pull up the suspension
into the Pasteur pipet. Add approx. 6 cc fixative to the tube. Place the pipet with
your cells into the fixative and carefully release the suspension in a slow steady
stream. Resuspend thoroughly with pipet. Refrigerate at least 30 minutes
(optional).
9. Spin down and wash with fixative at least two more times.
10. After last rinse, depending on size of pellet, leave approximately 0.5-1 cc of
fixative to make a cloudy suspension. You are now ready to make slides.
Alternate Protocols
1. If fewer cells than desired are in mitosis, you can try "synchronizing" the cells by
adding 50 µl colcemid to a T75 flask of cells. Leave the colcemid in for
approximately 2 hours (or more). Remove the supernatant containing the
colcemid. Rinse the flask gently with new medium and discard the rinse. Add fresh
medium to the flask and place back in the incubator until the next day. On day 2,
monitor the cells for optimal mitotic activity, usually 20-24 hrs after release of the
cells. Add the cell arrestors and harvest as usual.
2. If chromosomes are consistently too short using the standard colcemid protocol,
you can add to each T75 flask:
• 50 µl colcemid plus 15 µl Ethidium Bromide (EB) for 15 to 20 minutes.
• 200 µl Actinomycin D (final concentration of 2 µl/ml) and 200 µl Velban
(final concentration 0.01 µl/ml) for 30-60 minutes.
Harvest as usual, but increase the amount of time in KCl by at least 15 min.
AMD and EB have been shown to induce chromosome elongation or stretching,
which facilitates higher resolution G-banded chromosomes. However, AMD will
decrease the mitotic index of your harvest and may induce chromosome breaks.