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Cell cycle analysis: Synchronization of DT40 cells
George Zachos
University of Crete, Heraklion, Greece
Studies of the cell cycle often require synchronization of cell populations, i.e.
isolating cells at a specific cell cycle phase or stage
Methods of Cell Synchrony (I)• Chemical Methods
Agents block specific processes in the cell cycle and cause cells to arrest at particular stages
Thymidine prevents entry of cells into S-phase
Aphidicolin or Hydroxyurea arrest cells in S-phase
Nocodazole blocks cells in mitosis
Washing–off those agents would allow cells to resume cycling as synchronous population
Potential problem: It is difficult to ensure that there are no drug artifacts caused during cell synchronization that affect the experimental observations
Methods of Cell Synchrony (II)
• Physical Methods (No drugs)
Mitotic Shake-off. If adherent cell cultures are gently shaken, mitotic cells will get released into the medium.
Obviously this doesn’t work for DT40s!!
Cell Sorting. Direct selection of cells based on their DNA content using a fluorescence-activated cell sorter and an appropriate dye that binds DNA quantitatively in living cells.
This can be applied in DT40s but the capacity is very low
Elutriation. Isolating cells on the basis of their size
Cells become bigger as they move from G1 towards S, G2 and mitosis.
Using elutriation, any given cell culture can be fractionated into “small” (G1 ) cells, “medium” (S-phase) cells, “large” (G2 +M cells) and “very large” polyploid cells!
Elutriation -> size -> DNA content (cell cycle phase)
• Elutriation is ideal for suspension cells like DT40
• No drugs needed for synchronization
• Large amounts of cells can be fractionated (between 2 x107 and 1 x 109 cells)
• Cell fraction(s) of interest can be returned into culture and used for biochemical, cell cycle analysis, treated with drugs, etc.
Elutriation
The elutriator system
Elutriation medium reservoir
Injection of cell suspension
Pump
Pressure Gauge
Beckman centrifuge rotor
strobe light
viewing port
Collection flask
Elutriation chamber
Balance chamber
RPM
plastic tubing
plastic tubing
Elutriation of DT40s: Basic steps (I)
1. Setup cells
A lot of cells are needed! Typically we elutriate 2 x108 cells, i.e. the previous day we setup 10 x T150 flasks with 1 x 107 DT40 cells seeded in each flask
• It is essential that the cell population is maintained in the log phase of growth for at least 3-4 days prior to fractionation
• As a cell population reaches saturation, an increasing proportion of cells enters G0 . Those G0 cells have similar size to G1 cells and will fractionate together. Also, large number of G0 cells will decrease the yield of other (S-, or G2 -phase) cell fractions
• Cells might be damaged due to sub-optimal culture conditions (e.g. anoxia)
• If cells are too sparse, the total yield and percentage of S-phase cells will be lower than expected
2. Prepare mediumWe use approximately 4 lt of DT40 medium (sterile) per elutriation. Use RT medium for cells that will be cultured after fractionation. If you elutriate 2 or 3 cell lines prepare double, triple, etc the volume of medium!
Elutriation of DT40s: Basic steps (II)
Elutriation medium (for 1 lt)
10x DMEM (-Pyruvate, -Glutamine, -Bicarbonate, +NEAA) 100 mlFBS 100 mlHeat inactivated chicken serum 10 ml200 mM Glutamine 10 ml100 mM Sodium Pyruvate [#S8636 – Sigma] 10 ml7.5% Sodium Bicarbonate [S8761 – Sigma] 50 mlConc. HCl 500 μl1 M β-mercaptoethanol 10 μlPenicillin (6 mg/ ml) 5 mlStreptomycin (10 mg/ ml) 5 mldH2 O 700 ml
Elutriation of DT40s: Basic steps (III)
3. Prepare cells
• Pellet cells from the T150 flasks, at 1000 rpm for 5 min.
• Resuspend cells in 5 ml medium using a 5 ml pipette.
• Transfer cells in a syringe (without needle) for loading into the elutriator
5. Sterilize and prepare the system by passing
approx :
• 300 ml 70% ethanol
• 300 ml sterile H2 O
• DT40 medium
7. Slowly inject cell suspension
4. Start pump at 40 ml/ min
Collection flask
6. Start the rotor at high speed
(approx 3750 RPM)
Elutriation of DT40s: Basic steps (IV)
Medium continuously passing through the
system
Cells injected
Pump continuously running at 40 ml/ min
for the duration of the experiment Collection flask
Rotor running at high speed
(approx 3750 RPM)
Elutriation of DT40s: Basic steps (IV)
8. Allow approximately 300 ml of medium to run through the system
Cells are now inside the elutriation chamber!!
Cells suspended in medium enter chamber due to flow
stream
The elutriation process
centrifugal force = centrifugal speed (RPM) x mass
Sedimentation tendency of cells balanced by
counterflow
centrifugal g-force
counterflow stream
RPM
Further decrease centrifugal speed: bigger cells will also leave
the chamber
g-force
RPM
counterflow
Bigger cells will establish a new equilibrium
RPM
g-force counterflow
Decrease centrifugal speed: smaller cells will leave the
chamber
g-force
RPM
counterflow
Medium continuously passing through the
system
Cells injected
Pump continuously running at 40 ml/ min
for the duration of the experiment Collection flask
Rotor running at high speed
(approx 3750 RPM)
Elutriation of DT40s: Basic steps (V)
9. Decrease rotor speed (usually by 250 RPM steps). Allow 150 ml medium to run through. Smaller cell particles will be eluted and collected in sterile
flasks (300 ml falcon tubes)
10. Decrease rotor speed further (usually down to 1750
rpm) to get more fractions (usually 8). Collect fractions.
Elutriation of DT40s: Basic steps (VI)
11. After all fractions are collected, take a small aliquot from each and analyze by flow cytometry (propidium iodide, PI, staining):
• Pellet aliquot @ 1000 rpm, 5 min
• Resuspend cells in 500 μl PBS
• Fix with 5 ml 70% ethanol in PBS on ice for 30 min
• Spin down @ 1000 rpm for 5 min, resuspend pellet in 500 μl PBS + 20 μg/ ml Propidium Iodide + 250 μg/ ml RNAse A
12. Determine the PI profile of each fraction by flow cytometry
Total
Load 3750Fract
1
3500Fract
2
3250
Fract
3
3000
Fract
4
2750
Fract
5
2500Fract
6
2250
G1 fraction
Late S and G2 fraction
Fractionation of wt DT40 cells
Elutriation conditions are highly
reproducible! You should need to do
this pilot experiment only once!!
Flow rate: 40 ml/ min
Total
Load 3500
Fract
1
3250
Fract
2
3000
Fract
3
2750
Fract
4
2500
Fract
5
2250
Fract
6
2000
G1 fraction
Late S and G2 fraction
Fractionation of Chk1-/-
DT40 cells
Flow rate: 40 ml/ min
Each cell line has distinct elutriation profile depending on the cell size
G1 fraction
DT40 Chk1-/-
3250 rpm
2750 rpm
2750 rpm
2000 rpm
Flow rate: 40 ml/ min
G2 fraction
Elutriation of DT40s: Basic steps (VII)
13. The desired cell fraction(s) can now be manipulated as required and returned to the incubator for experimental analysis!
Example: Using elutriated cells to investigate the role of Chk1 protein kinase
in the mitotic
spindle checkpoint
Chk1-/-
cells fail to sustain mitotic arrest in the presence of taxol
DT40
Chk1-/-
020406080
100
taxol nocodazoleFrac
tion
of c
ells
that
re
mai
ned
in m
itosi
s (%
) Mitotic arrest (time-lapse)
0 8 12 16 20 0 8 12 16 20 0 8 12 16 20hrs in taxol
H1
DT40 Chk1-/- Rev
Cdc2 kinase activity
Mitotic Index
Time in taxol (hrs)
pH3
posi
tive
(%)
DT40
Chk1-/-
Rev
0
20
40
60
80
0 4 8 12 16 20
• We elutriated DT40 and Chk1-/- cells at predetermined conditions
• Isolated G2 cell fractions were returned into culture in the presence of spindle drugs for several hours
• Flow cytometry, biochemistry, confocal microscopy analysis performed
Elutriation of DT40s: Basic steps (VIII)
14. Don’t forget to clean the elutriator after use:
• Empty medium
• Sterilize by passing 300 ml H2 O followed by 300 ml 70% ethanol through the system
• Dismantle and clean the elutriation chamber!
Technical Factors
Rotor
The Beckman JE-6B rotor runs the standard-size elutriation chambers. The JE-6B rotor is capable of speeds up to 6000 rpm and can be run in any Beckman centrifuge. The JE-5.0 rotor can also run the large-volume chambers but requires a bigger centrifuge (Beckman J-6).
Chamber
The standard chamber (4.5 ml) can fractionate between 2 X107 and 1 X 109
cells (in our hands that’s plenty). The larger 40 ml version has ten times the capacity.
Pump
Nearly any peristaltic pump with flow rates (2-100 ml/ min) may be used (we normally operate it at 40 ml/ min). Minimal pulsation is desired to maintain a steady flow of elutriation medium.