Home › Microbiology › Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes
Microbiology JoVE (Open Access) Citable · DOI

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

DOI: 10.3791/57215-v
What you'll learn
  • ✓ Synchronize RPE-1 hTERT cells using serum starvation and nocodazole arrest
  • ✓ Generate aneuploid cells by inhibiting Mps1 kinase activity during mitosis
  • ✓ Isolate cell cycle-arrested cells with complex karyotypes (ArCK) from aneuploid populations
  • ✓ Characterize karyotype complexity and cell cycle status of enriched ArCK cells
Protocol

Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

Difficulty
advanced
Total time
~5–7 days (cell synchronization, aneuploidy induction, selection, and characterization)
Model organism
RPE-1 hTERT
Biosafety
BSL-1

Steps

1
Synchronize RPE-1 hTERT cells at G1/S boundary

Arrest cells using serum starvation followed by nocodazole treatment to synchronize the population at a defined cell cycle stage before aneuploidy induction.

▶ 00:32
2
Generate aneuploid cells by Mps1 inhibition

Inhibit Mps1 kinase activity during mitosis to disrupt checkpoint control and generate cells with abnormal chromosome numbers. Remove cycling aneuploid cells to enrich the arrested population.

▶ 01:34
3
Isolate and characterize ArCK cells in vitro

Perform karyotyping and cell cycle analysis to confirm isolation of aneuploid cells with complex karyotypes (ArCK) that have undergone cell cycle arrest.

▶ 03:36
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