synchronization of cell lines

William Meikrantz meikrant at husc7.harvard.edu
Tue Apr 25 10:15:25 EST 1995


In our own work, 
mostly with transformed epithelial cells or carcinoma-derived cells, we 
have found the following treatments to be well tolerated:

1. Synchrony in S phase by adding high concentrations of thymidine to the 
medium.  Concentrations from 2.5 to 25 mM can be used.  High thymidine 
has a negative feedback effect on nucleotide biosynthesis; without a 
proper supply of nucleotides, DNA replication comes grinding to a halt, 
leaving the cell stranded in S phase.  Overnight incubation will yield a 
population that is arrested throughout S phase.  

The double thymidine block provides a more precisely synchronized
population: after overnight incubation, replace with fresh medium and
allow the cells to recover.  Waiting 8-10 h will ensure that all the cells 
have moved out of S phase.  Then the high thymidine block is imposed 
again--this time, since there are no cells already IN S Phase, a uniform 
early S phase population obtains.  Again, 12 h - overnight is 
sufficient.  

The extent of arrest is conveniently monitored, if there is no FACS 
handy, simply by doing a mitotic index determination on a sample of the 
cells.

Other S phase arrest agents, like hydroxyurea (1.0 - 2.5 mM; we use 2.5 
mM), tend to be less reversible and show significant toxicity after > 16 
h.  The DNA polymerase inhibitor, aphidicolin, is reportedly quite 
reversible (5 microgram/ml is a typical concentration) with low 
cytotoxicity.  

Double thymidine block followed by release into fresh medium results in a 
population that moves synchronously through S phase, G2 and M.  The 
synchrony begins to break down during the subsequent G1 phase.

2. Synchrony of mitotics.  We have found low concentrations (10-40 ng/ml) 
of the antimicrotubule agent nocodazole to be effective, reversible, and 
non-toxic as long as the time of incubation is minimized (kept to less 
than 16 h).  Prolonged exposure leads to apoptosis.  Agents such as the 
vinca alkaloids and colchicine and colcemid are as effective but less 
reversible.  Colcemid is available in a tissue culture formulation from 
Gibco.

Mitotics are conveniently separated from adherent monolayers by gentle 
(GENTLE!) shaking or tapping of the flask or dish, or by gently washing 
with warm medium.  This provides a way of obtaining a synchronous 
population without chemical treatment: mitotics can be shaken off an 
asynchronously growing monolayer culture, then held in mitosis by placing 
them on ice.  Once a sufficient number of cells have been collected, 
plate in warm medium.  The cells will adhere within 1-3 h and proceed 
synchronously through G1 into S phase.

Other methods of synchrony, by treatment with lovastatin, mimosine, 
deferoxamine, dibutyryl cAMP, etc. tend to vary in effect from cell type 
to cell type, with regard to effectiveness, point of arrest, and degree 
of toxicity.  Most cells will arrest at the G1-S boundary in the 
presence of cycloheximide or actinomycin D; this is frequently toxic, 
however.  

Because cells of myeloid or lymphoid lineages are often physiologically
poised to undergo apoptosis at the slightest perturbation, synchronization
of either primary or transformed cells can be tricky.  Hybridomas appear
near-impossible to synchronize, and the susceptibility of many leukemia or
lymphoma-derived lines to cell cycle arrest agents is precisely why these
malignancies are generally good responders to chemotherapy.  However,
while suspension growth makes mitotic shake-off impossible as a
synchronization method, it does permit removal of cells that didn't
survive synchronization by whatever chemical method by Ficoll.  (Care must
be taken here that a resistant subpopulation is not being selected for.) A
good account of cell cycle arrest methods in a lymphoid line (mature,
non-transformed CD4+ and CD8+ lymphocytes) can be found in Boehme, S.A. 
and M.J. Lenardo, Eur. J. Immunol. 23:1552-1560 (1993), where a variety of
synchronizing agents were used successfully. 


Hope this is helpful!

Bill Meikrantz
Molecular and Cellular Toxicology
Harvard School of Public Health






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