Monitoring mitotic cell division in DT40 cells - Events

Tubulin DNA
Monitoring
mitotic cell
division in DT40
cells

Our scientific goal: To investigate the
role of Chk1 in chromosome
segregation during mitotic cell division
• Monitor chromosome segregation during mitotic cell division
in living wild-type (wt) and Chk1-/- DT40 cells by time-lapse
video microscopy
• With time-lapse video microscopy we can monitor events in
living cells as they happen!

Time-lapse video microscopy
• Cells grow in appropriate cell container (e.g. 6-well plate or 30 mm petri dish)
at controlled temperature and CO 2 conditions and are monitored by light
microscopy (phase contrast or DIC)
• If cells express a fluorescence protein of interest they can also be monitored
by fluorescence microscopy
• Cells are monitored for periods up to several hours and a digital photo of
them is taken at desired intervals (e.g. every two minutes, 30 sec, etc)
• If desired, both a fluorescence and a phase contrast/ DIC photo can be taken
at every interval
• At the end of the experiment, part or the whole series of still images can be
compressed into a movie

specimen
Components of a time-lapse video
microscopy system: the microscope
Zeiss, Axionert 200
halogen lamp
mercury
lamp
shutter
objective lenses
• Inverted fluorescence microscope (for
living cells)
• Filters for FITC fluorescence
• Objective lenses for DIC or phase
contrast
• Magnification of objective lenses: 4X,
10X, 20X, 40X

Components of a time-lapse video
microscopy system: the temperature
control module
Temperature control module
for 30 mm dishes

Components of a time-lapse video
microscopy system: the environmental
chamber
The environmental
chamber is connected to
a CO 2 source to maintain
appropriate CO 2
conditions (this is
important when cells are
monitored for several
hours)

Components of a time-lapse video
microscopy system: digital camera,
automatic shutter and light beams
• The digital camera is programmed
through a control unit to take photos of the
specimen at predetermined intervals for
the period of the experiment
• The microscope shutter automatically
switches from light to fluorescence images
• Both mercury and halogen light beams
automatically switch themselves on before
photos are taken and then switch
themselves off to minimally interfere with
the specimen and avoid photobleaching of
fluorescent molecules
High speed Digital
CCD camera

CO 2 and
temperature
control unit
Overall components of a time-lapse
video microscopy system
Environmental chamber

Generation of DT40 cells stably
expressing H2B:GFP protein
• To monitor chromosome segregation, we generated stable clones of wt
and Chk1-/- DT40 cells expressing the chromatin marker histone 2B fused
to GFP (H2B:GFP protein)
• Chromosomes in those cells can be detected by fluorescence microscopy
(green fluorescence)
General Steps:
1. Obtain plasmid coding for H2B:GFP
2. Electroporate plasmid into cells
3. Select individual clones expressing the plasmid (green clones!)
4. Use clones in time-lapse microscopy experiments

Plasmid coding for H2B:GFP protein
Ori
CMV
Promoter
GFP
H2B:GFP
vector
Neomycin
Resistance
H2B
Poly-A
Kan R

Electroporation of DT40 cells (I)
If using the Amaxa nucleofector system, follow the manufacturer’s
instructions!!
Materials and apparatus
• 25 μg of plasmid per 5 x 10 6 cells. (We only linearize plasmid for gene
targeting experiments)
• Optimem medium (from Gibco BRL)
• 4 mm electroporation cuvettes (from Flowgen)
• Biorad electroporation micropulser (or similar)

Method
Electroporation of DT40 cells (II)
• Use 5 x 10 6 cells. Cells must be in the log growth phase and I always split them
approx 1:4 the day before electroporation
• On the day of electroporation count cells and spin down 5 x 10 6 cells. Resuspend
cells in 1ml of optimem + 1% DMSO. Add plasmid DNA and put the cells + DNA
into an electroporation cuvette
• Electroporate at 300 volts, 960μF
• Dilute cells into 20 ml DT40 growth medium and incubate O/N at 39.5 o C, 5%
CO 2

Isolation of DT40 clones
• The next day, take the volume up to 140 ml with DT40 growth medium
containing the required selection agent (e.g. G418 at 1.5 mg/ ml) and 50 µg/ ml
amphotericin (fungicide)
• Split the 140 ml between 7 x 96 well plates using a multi-channel pipette (200
μl per well). Incubate at 39.5 o C, 5% CO 2
• After approx 3 weeks examine the wells for small colonies (balls) of antibiotic
resistant cells. Clones expressing H2B:GFP exhibit green fluorescence and are
easily identified!
• Transfer a few clones into 24 well plates and progressively expand them
• Verify DNA fluorescence and that the stages of mitosis look OK by confocal
microscopy. Choose the appropriate clone(s)

Setting up DT40s for time-lapse
microscopy (I)
Prepare coverslips
• Coat 12 mm round coverslips with poly-L-lysine, 10 μg/ ml in sterile PBS, O/N,
at 4 o C
• Wash a couple of times with PBS: coverslips are ready to use. You can keep
coated coverslips in PBS at 4 o C for a few days
• If in a hurry, you can also coat coverslips at 37 o C for 2 hrs and then wash in
PBS and use

Set up cells
Setting up DT40s for time-lapse
microscopy (II)
• On the previous day, count cells expressing H2B:GFP, spin them down and
resuspend in fresh medium at approximately 1 x10 5 cells/ ml
• Place a poly-L-lysine-coated coverslip inside an empty 30 mm petri dish
• Drop 100 μl (i.e. 1 x 10 4 ) cells onto the coverslip (you don’t want cells too crowded!!)
• Leave at room temperature for approximately 1h for DT40s to sit down
• Carefully put 2 ml growth medium inside the 30 mm dish and return to the incubator
• Next day, monitor cells on the time-lapse microscope and get still photos or movies!!

Monitoring mitotic cell division by time-
lapse microscopy (I)
Photo 1
H2B:GFP DIC
Photo 2
H2B:GFP DIC
Photo 3
H2B:GFP DIC
Photo 4
H2B:GFP DIC

Monitoring mitotic cell division by time-
lapse microscopy (II)
H2B:GFP
Photo 5
Photo 6
DIC
H2B:GFP DIC
Photo 7
H2B:GFP DIC
Photo 8
H2B:GFP DIC

Monitoring mitotic cell division by time-
lapse video microscopy
DNA DIC

metaphase
anaphase
Normal chromosome
segregation
spindle pole
sister chromatids
kinetochore
metaphase
anaphase
Chromosome missegregation
(polar
chromosome)

Chk1-deficient Chk1 deficient DT40 cells exhibit high
frequency of “polar polar” chromosomes
wt DT40 Chk1-/-
Normal anaphase Anaphase with “polar” chromosome
Green: DNA

Chk1-deficient Chk1 deficient DT40 cells exhibit high
frequency of anaphase bridges
DNA
DNA
Chk1-/-
Chk1-/-
Tubulin DNA
Tubulin DNA
Chk1-/-
Anaphase bridge
Green: DNA

Chk1-deficient Chk1 deficient DT40 cells exhibit high
frequency of cytokinesis failure
Chk1-/-
Failed cytokinesis
Green: DNA

Living
cell
Apoptotic
cell
Mitotic failure can lead to cell death
Electron microscopy
Cell death after mitotic failure
Time-lapse microscopy
Green: DNA