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Preparation and Staining of Mammal Cells to View Sister Chromatids

By Charles E. Geach



Type of Activity:

  • Laboratory

Target Audience:

  • Biology
  • AP Biology


Abstract

This activity was developed in the laboratory of Dr. Pablo Arenaz at the University of Texas at El Paso during a summer teacher research fellowship program sponsored by the American Society For Cell Biology. This protocol allows you to see chromatids in mammal tissue. The protocol below was used to observe sister chromatid exchanges in Chinese Hamster Ovary Cells (Cell line AA8).

Background Information

Notes for teacher:

This procedure took place in a state of the art cell tissue culture facility, however, the hamster cells used are easily grown and can be grown in a common incubator. The other procedures, as well, may be performed in a basic high school lab. It is a lengthy protocol but it produces slides of mammalian chromosomes that are easily observed under a good high power microscope lens and worth the effort for those wanting to observe mammalian chromosomes.

Objective

To culture and stain mammal cells allowing observation of sister chromatids.

Materials needed:

Mammal CellsTissue Culture Flasks
DMEMCulture Dishes
Newborn Calf SerumL-glutamine
PenicillinIncubator
BrduColcemid
Rubber PolicemanCentrifuge
KClWater bath
MethanolGlacial Acetic Acid
Pasteur PipetGlass Slides
Pipet15 ml Centrifuge tube
Bunsen BurnerGiemsa Stain


Protocol:

  1. Chinese Hamster Ovary Cells (Cell Line AA8) were cultured in complete media using:
    DMEM (Dulbecco's modified Eagles medium), 10%(v/v)Newborn Calf Serum, 2mM L-glutamine, and 100 U penicillin. The cells were grown in plastic tissue culture flasks placed in an incubator at 37°C and 5% CO2. Cells were grown until they reached 5x105 cells /ml. (Approx. 5 days)

  2. Cells are seeded onto culture dishes at 5x105 cells/ml and incubated for 24-48 hours at 37°C with 5% CO2.

  3. To the cultures are added Brdu (Bromodeoxyuridine) at 30uM (30 ul of a 1mM solution/10 ml media) and cells are again incubated for an additional 24-48 hours depending on cell type. This differentially labels the sister chromatids

  4. Two hours prior to harvesting, colcemid is added to each culture at 0.5 ml/10ml of media. This arrests cells at metaphase.

  5. Cells are harvested by scraping using a rubber policeman into 15 ml centrifuge tubes.

  6. Spin tubes at 3,000 rpm for 10 min. , remove the supernatant except for approximately .5ml and do not disturb the pellet of cells.

  7. Resuspend pellet in remaining solution and add 5 ml. of 0.075 KCl at 37°C. Place in a water bath at 37°C for 5 min. *( Do not let cells stay in this hypotonic solution for more than 16-18 min.)

  8. Spin tubes at 3,000 rpm for 6 min. , aspirate off KCl except for .5 ml. resuspend and add 5 ml. of 100% methanol ( fixative) to each tube and let sit 2 min.

  9. Spin at 3,000 rpm for 10 min. and aspirate off supernatant except for .5 ml., resuspend and add 5 ml. ice cold 3:1 methanol:acetic acid and let sit 10 min.

  10. Spin at 3,000 rpm for 10 min. and aspirate all but .5 ml. and let sit in ice bath.

  11. Resuspend using a pasture pipet and drop onto cold wet slides. The slides are flamed and allowed to air dry for 24 hours.

  12. Slides may be stained with Giemsa stain (3% Giemsa in phosphate buffer for 10 min.) to see chromosomes.
    Differential staining with fluorescence -Hoechst (bisbenzamide ) and Giemsa for observing sister chromatid exchanges may be done instead. Slides are exposed to UV light for 30 min. after staining with Hoechst and subsequent staining with 10% Giemsa.


Reference:

Arenaz, P., L. Bitticks, K.H. Pannell and S. Garcia
Genotoxic potential of crown ethers in mammalian cells: Induction of sister-chromatid exchanges, Mutation Research,280 (1992) 109-115

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