Materials and Reagents

Cell lines: FaDu (ATCC, catalog number: HTB-43^TM )
Trypsine (Life Technologies, Gibco^®, catalog number: 15400-054 )
10% fetal bovine serum (Thermo Fisher Scientific, catalog number: SH30071.03 )
1% Penicillin-Streptomycin (10,000 U/ml; 10,000 μg/ml) (Life Technologies, Gibco^®, catalog number: 15140-122 )
Colcemid (10 μg/ml) (Life Technologies, Gibco^®, catalog number: 15212-012 )
Hypotonic solution (0.075 M KCl) (J.T.Baker^®, catalog number: 7447-40-7 )
Glacial acetic acid (J.T.Baker^®, catalog number: 64-19-7 )
Fixative (see Recipes)
Growth medium (see Recipes)

Equipment

10 cm cell culture dishes (Trueline Valve Corpotation, catalog number: TR4002 )
Centrifuge tube
Microscope (OLYMPUS, model: DP71 )
CO₂ incubator
Centrifuge at room temperature (using “relative centrifugal force, rcf”)
Water bath

Procedure

Day 1

Seed 5 x 10⁵ cells on 10 cm cell culture dishes for attachment.
Cells were incubated at 37 °C, 5% CO₂ for 48 h.

Day 3

Add 0.1 ml colcemid, which can collapse mitotic spindles and prevent the completion of mitosis, to each dish and mix gently. Incubated at 37 °C, 5 % CO₂ for 2 h.
Transfer medium to centrifuge tubes from the cell culture dishes. Use PBS to wash the dishes, and remove PBS. Then, add 1 ml 0.1% Trypsine into the dishes at 37 °C, 5% CO₂ for 2 min. Also, transfer the mixture (Trypsine and cells) into the centrifuge tubes and mix with the medium which is transferred to centrifuge tubes before we use PBS to wash the dishes. Further, centrifuge at 100 rcf for 10 min at room temperature.
Discard the supernatant and leave 0.5 ml medium to mix the pellet gently.
Resuspend the pellet in 5-7 ml 37 °C hypotonic solution and mix thoroughly. Incubate in water bath at 37 °C for 10 min.
Centrifuge at 100 rcf for 10 min at room temperature.
Discard the supernatant and leave 0.5 ml solution to mix the pellet gently.
Resuspend the pellet in 5 ml cold fixative (drop by drop and flick the tube between drops to prevent cell clumping).
Put the centrifuge tube on ice at least 20 min.
Centrifuge at 100 rcf for 10 min at room temperature.
Discard the supernatant and add 3-5 ml cold fixative.
Repeat steps 9-10 three times or until the supernatant is clear and the pellet become white.
After the final centrifugation, suspend the cells in a few drops of cold fixative to give a slightly opaque suspension.
Drop 1-2 drops onto wet and clean slides (Before that, the slides have to be rinsed by 1-2 drops of cold fixative.)
Dry the slides at room temperature.
Observe the chromosomes with the microscope (Olympus DP71 with 200x magnification).
Count the number of chromosomes about 50 cells (A normal human cell have 23 pairs of chromosomes.).
Note: Chromosomes center in a scope, we define that “one cell”.

Representative data

Figure 1. Representative pictures of karyotype analysis in FaDu cell line (see Chou et al., 2013)

Recipes

Fixative
Methanol and glacial acetic acid (3:1) to be made fresh and chilled before using
Growth medium
RPMI 1640 supplemented with:
10% fetal bovine serum
1% Penicillin Streptomycin (10,000 U/ml; 10,000 μg/ml)

Acknowledgments

This protocol has been adapted from Chou et al. (2013).

References

Chou, C. H., Yang, N. K., Liu, T. Y., Tai, S. K., Hsu, D. S., Chen, Y. W., Chen, Y. J., Chang, C. C., Tzeng, C. H. and Yang, M. H. (2013). Chromosome instability modulated by BMI1-AURKA signaling drives progression in head and neck cancer. Cancer Res 73(2): 953-966.