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

G1

G2

S

M & cytokinesis

The eukaryotic cell cycle

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: A powerful tool for synchronizing DT40 cells

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.