Preparation and Staining of Mammal Cells to View Sister Chromatids

By Charles E. Geach

Type of Activity:

• Laboratory

Target Audience:

• Biology
• AP Biology

Abstract

Background Information

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

Materials needed:

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:

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