Chromosome Spread and Telomere FISH Analysis

Mitotic cells were collected either by shake-off or by colcemid (50 ng/mL) enrichment for 3 h^{11 (link),35 (link)}. Cells were incubated in 0.075 M KCl followed by an overnight fixation in methanol/acetic acid (3:1). Chromosome spreads were generated by dropping cells onto −80 °C precooled glass slides. Slides were next stained in Giemsa staining solution (Sigma) for 4 min. Stained slides were analyzed for sister chromosome separation by Nikon Eclipse 80i microscope^{11 (link)}. Spreads without Giemsa staining were used for telomere FISH. Telomere FISH was performed as detailed above using TAMRA-OO-[CCCTAA]3 labeled PNA probe (PANAGENE, Cat. no. F2001)^{36 (link)}.

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Zhao B., Lin J., Rong L., Wu S., Deng Z., Fatkhutdinov N., Zundell J., Fukumoto T., Liu Q., Kossenkov A., Jean S., Cadungog M.G., Borowsky M.E., Drapkin R., Lieberman P.M., Abate-Shen C.T, & Zhang R. (2019). ARID1A promotes genomic stability through protecting telomere cohesion. Nature Communications, 10, 4067.

Publication 2019

Giemsa staining solution Eclipse 80i microscope TAMRA-OO-[CCCTAA]3 labeled PNA probe

Acetic acid Cells Chromosome Colcemid Fish Giemsa Methanol Shake Telomere

Corresponding Organization :

Other organizations : The Wistar Institute, Columbia University Irving Medical Center, University of Pennsylvania

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Variable analysis

independent variables

Method of mitotic cell collection (shake-off or colcemid enrichment)

dependent variables

Sister chromosome separation
Telomere FISH

control variables

Concentration of colcemid (50 ng/mL)
Duration of colcemid treatment (3 h)
KCl incubation
Overnight fixation in methanol/acetic acid (3:1)
Chromosome spreading by dropping cells onto -80 °C precooled glass slides
Giemsa staining for 4 min
Microscope used for analysis (Nikon Eclipse 80i)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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