Przejdź do zawartości
Merck

Release from cell cycle arrest with Cdk4/6 inhibitors generates highly synchronized cell cycle progression in human cell culture.

Open biology (2020-10-15)
Eleanor Wendy Trotter, Iain Michael Hagan
ABSTRAKT

Each approach used to synchronize cell cycle progression of human cell lines presents a unique set of challenges. Induction synchrony with agents that transiently block progression through key cell cycle stages are popular, but change stoichiometries of cell cycle regulators, invoke compensatory changes in growth rate and, for DNA replication inhibitors, damage DNA. The production, replacement or manipulation of a target molecule must be exceptionally rapid if the interpretation of phenotypes in the cycle under study is to remain independent of impacts upon progression through the preceding cycle. We show how these challenges are avoided by exploiting the ability of the Cdk4/6 inhibitors, palbociclib, ribociclib and abemaciclib to arrest cell cycle progression at the natural control point for cell cycle commitment: the restriction point. After previous work found no change in the coupling of growth and division during recovery from CDK4/6 inhibition, we find high degrees of synchrony in cell cycle progression. Although we validate CDK4/6 induction synchronization with hTERT-RPE-1, A549, THP1 and H1299, it is effective in other lines and avoids the DNA damage that accompanies synchronization by thymidine block/release. Competence to return to cycle after 72 h arrest enables out of cycle target induction/manipulation, without impacting upon preceding cycles.

MATERIAŁY
Numer produktu
Marka
Opis produktu

Sigma-Aldrich
Insulin solution human, sterile-filtered, BioXtra, suitable for cell culture
Sigma-Aldrich
hEGF, EGF, recombinant, expressed in E. coli, lyophilized powder, suitable for cell culture
Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium - high glucose, With 4500 mg/L glucose, sodium pyruvate, and sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Thymidine, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Anti-Rabbit IgG (whole molecule)–Alkaline Phosphatase antibody produced in goat, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Nocodazole, ≥99% (TLC), powder
Sigma-Aldrich
Hydrocortisone, BioReagent, suitable for cell culture
Sigma-Aldrich
Propidium iodide, ≥94.0% (HPLC)
Sigma-Aldrich
Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301, clone JBW301, Upstate®, from mouse
Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium/Nutrient Mixture F-12 Ham, With 15 mM HEPES and sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
HOE 140, ≥94%
Sigma-Aldrich
Anti-Mouse IgG (whole molecule)−Alkaline Phosphatase antibody produced in goat, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Anti-KIF11 antibody produced in rabbit, affinity isolated antibody
Sigma-Aldrich
5-Bromo-4-chloro-3-indolyl phosphate disodium salt, ≥98% (HPLC)