Synchronization of Cells

Method 18
SYNCHRONIZATION OF CELLS
Eiichiro Sonoda
Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe,
Kyoto 606-8501, Japan
Abstract: Synchronization of cells is essential to study cell cycle specific events. If,
for example, one suspects that a given DNA repair pathway is used in a
particular cell cycle phase, the protocol can be used to enrich cells in each
phase of the cell cycle and analyze the cellular response to DNA damage.
Synchronization is also useful, when a gene is essential for a particular
phase of the cell cycle. If a gene is, for example, essential for mitosis,
synchronization of the cells in G1 phase with concomitant inactivation of
the gene enables us to study the function of the gene in interphase, and to
follow synchronous cell cycle progression to M phase. Two synchronization
methods: centrifugal elutriation to enrich
n G1, S or G2 phase cells and
nocodazole-mimocine sequential treatmentt to enrich cells at the G1/S
boundary are described. Centrifugal elutriation
t can be achieved in less time
(0.5–2 h) and with very little physiological stress to the cells whereas
synchronization by drugs, such as nocodazole and mimocine, may result in
unfavorable side effects.
Key words: Protocol, synchronization.
Materials
1. SAMPLE
DT40 cells (1~10 x 107) in culture.
415
JJ.-M.
M Buers
r tedde and S. Take
k da (eds.), Reviews andd Protocols in DT40
T Research, 415–418.
© 2006 Springer.
416 Method 18
2.1 FOR ELUTRIATION
Reagents
DT40 cell culture medium
Elutriation buffer (0.01%EDTA/1%FBS/PBS, filtrated with 0.22um
filter); 2~3L
70% ethanol; 0.5L
Sterilized water; 1L
Propidium iodide
Ribonuclease (RNase)
Equipment
Elutriator (Hitachi Koki R5E system: 4.7ml Separation chamber;
equivalent to Beckman JE-5.0 System)
Flowcytometry
2.2 FOR NOCODAZOLE-MIMOCINE BLOCK

Reagents
DT40 cell culture medium
Nocodazole (10 mg/ml in DMSO, stock at –20°C)
L-Mimocine (100mM in acidic water, stock at –20°C)
3.1 Method for Elutriation
1) Assemble the elutriator rotor, elutriator chamber and the elutriator

centrifuge according to manufacturers’ directions.
2) Sterilize the apparatus by running 70% ethanol at 20mL/min for
30min, and then sterilized water for 30min without turning on the
centrifuge.
3) Replace the water with the elutriation buffer and run the centrifuge at
2,000 r.p.m. at 4°C.
4) Calibrate the speed of pump and the actual flow rate.
5) Harvest 5~10 x 107 cells and resuspend in 3~5ml of ice-cold elutriation
buffer. Pass through nylon mesh to remove clumped cells. Maintain
the cells on ice.
6) Inject the cells into a sample reservoir and then load them into the
separation chamber. Start with a flow rate of 10mL/min. Increase the
flow late incrementally and collect the cell fraction serially as follows;
18. Synchronization of Cells 417
Fraction 1:10mL/min, 200ml

7) Concentrate the cells by low-speed centrifugation (1.200 r.p.m. for
5min) at 4°C. Resuspend each fraction in 10ml of ice-cold elutriation
buffer and count the numbers of cells. Keep the cells on ice to prevent
cell cycle progression.
8) To determine the purity of cells, take a small aliquot from each
fraction (~1 x 105) and spin down. Fix the cells with 70% ethanol for
5~10 min and resupsend the cells in PBS containing 5µg/ml of
propidium iodide and 100µg/ml of RNAse. Analyze the content of
DNA by flowcytometry. In most cases, cells in G1, S and G2/M are
enriched in the fraction 1~2, 3~5 and 6~7, respectively.
3.2 METHOD FOR NOCODAZOLE-MIMOCINE

BLOCK
1) Add nocodazole to the cell culture at a final concentration of 0.5
µg/ml and incubate for 4 hr.
2) Wash the cells three times with warm medium, and incubated with
medium containing 0.8 mM mimosine for 12 hours.
3) Wash the cells three times with warm medium and release them
from the G1/S block.
4. Troubleshooting
1)Poor enrichment of synchronized population; Before cell separation, it

is essential to calibrate the actual flow rate and the speed of pump.
Measure the actual flow rate with measuring cylinder when the speed
of rotation becomes constant.
2)Low recovery of cell; In DT40 cells, the percentages of cells in G1, S
and G2 phase is 10~15, 60~70 and 15~20%, respectively, in an
asynchronous population. Accordingly, the number of the G1-enriched
cells is small, i.e. around 1~5 x 106 from 5 x 107 asynchronous cells. In
addition, the quality of the sample cell culture is critical and one
418 Method 18
should use exponential growing cells of high viability. Before

elutriation, cells should be resuspended completely in the elutriation
buffer to avoid clogging.
3) Low viability of cells in mimosine-nocodazole method; Since
mimosine is toxic to cell culture, longer exposure of cells to mimocine
causes massive cell death. Do not treat cell with mimosine longer than
12 hours.
4) Poor synchronization of the cell in mimosine-nocodazole method;
Residual drugs often cause aberrant cell cycle progression. Do not use
cold medium or PBS for washing.

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Method 18

Key words: Protocol, synchronization.

DT40 cells (1~10 x 107) in culture.

2.1 FOR ELUTRIATION

2.2 FOR NOCODAZOLE-MIMOCINE BLOCK

3.1 Method for Elutriation

1) Assemble the elutriator rotor, elutriator chamber and the elutriator

Fraction 1:10mL/min, 200ml

3.2 METHOD FOR NOCODAZOLE-MIMOCINE

1)Poor enrichment of synchronized population; Before cell separation, it

should use exponential growing cells of high viability. Before

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