that filopodia can be identified and selected from single cells. If you
are interested in isolating TNTs, then the cells need to be in closer
proximity in order to be able to form TNTs and therefore a higher
cell density is required. These numbers must be predetermined for
each cell types used, as well as for the types of cellular protrusion of
interest.
1. Pre-warm a 50-mL centrifuge tube with CAD media in the
37
C water bath. Place 3 mL of media into a 15-mL conical
tube and 5 mL of media into a T25 plate. Remove the cells
from the liquid nitrogen tank. Thaw the cryotube with CAD
cells in the 37
C water bath. When almost thawed (it is acceptable for some ice crystals to be left), take the tube out of the
water bath and move to the tissue culture hood. As freezing
media contains DMSO, which is toxic for cells, these steps have
to be done quickly.
2. Add 1 mL of media to the cells (approximately 1,500,000
cells), resuspend them, and transfer them into the prepared
15-mL conical tube. Spin the conical tube in a centrifuge at
1000 Â g for 4 min (the cells will collect at the bottom as a
pellet). Carefully aspirate and discard the supernatant from the
15-mL conical tube and add 1 mL of media to the cell pellet
and resuspend gently. Then, pull up all of the cell mixture into
the pipette and transfer it to the prepared T25 plate. Look at
cells before placing them in the incubator. The cells should be
round and shiny.
3. Next day, check that the cells have adhered to the plate and are
healthy and nondifferentiated and look at their confluency.
Grow cells until they become 80% confluent (usually 2 days).
4. To split the cells, remove the media and dead cells, wash once
with PBS, and add 1 mL of media. Tap the flask to detach the
cells (mechanical dissociation) and observe under the microscope to ensure that the majority of the cells are detached. Add
3 mL of fresh media along the walls to scrape out all the cells.
Pipette out the total 5 mL of the cultures, by pressing the tip of
the pipette on the bottom of the flask to break clumps and
check under the microscope to ensure that you have mostly
single cells. Add 1 mL of the culture (approximately 1,000,000
cells) to the new flask and take the volume of fresh media
needed to make up the new flask to 6 mL.
5. Alternatively, cells can be grown in a 60- or 10-cm culture dish.
In this case, cells can be detached mechanically by pipetting
them up and down, rather than tapping the flask.
6. After 2 days, cells should be 80% confluent, and they can be
split again. Cells should be split a minimum of two times before
seeding them in an MMI live chamber dish to ensure cell
viability. Once the cells have been split two times, they can be
30
Ana Gordon and Karine Gousset
are interested in isolating TNTs, then the cells need to be in closer
proximity in order to be able to form TNTs and therefore a higher
cell density is required. These numbers must be predetermined for
each cell types used, as well as for the types of cellular protrusion of
interest.
1. Pre-warm a 50-mL centrifuge tube with CAD media in the
37
C water bath. Place 3 mL of media into a 15-mL conical
tube and 5 mL of media into a T25 plate. Remove the cells
from the liquid nitrogen tank. Thaw the cryotube with CAD
cells in the 37
C water bath. When almost thawed (it is acceptable for some ice crystals to be left), take the tube out of the
water bath and move to the tissue culture hood. As freezing
media contains DMSO, which is toxic for cells, these steps have
to be done quickly.
2. Add 1 mL of media to the cells (approximately 1,500,000
cells), resuspend them, and transfer them into the prepared
15-mL conical tube. Spin the conical tube in a centrifuge at
1000 Â g for 4 min (the cells will collect at the bottom as a
pellet). Carefully aspirate and discard the supernatant from the
15-mL conical tube and add 1 mL of media to the cell pellet
and resuspend gently. Then, pull up all of the cell mixture into
the pipette and transfer it to the prepared T25 plate. Look at
cells before placing them in the incubator. The cells should be
round and shiny.
3. Next day, check that the cells have adhered to the plate and are
healthy and nondifferentiated and look at their confluency.
Grow cells until they become 80% confluent (usually 2 days).
4. To split the cells, remove the media and dead cells, wash once
with PBS, and add 1 mL of media. Tap the flask to detach the
cells (mechanical dissociation) and observe under the microscope to ensure that the majority of the cells are detached. Add
3 mL of fresh media along the walls to scrape out all the cells.
Pipette out the total 5 mL of the cultures, by pressing the tip of
the pipette on the bottom of the flask to break clumps and
check under the microscope to ensure that you have mostly
single cells. Add 1 mL of the culture (approximately 1,000,000
cells) to the new flask and take the volume of fresh media
needed to make up the new flask to 6 mL.
5. Alternatively, cells can be grown in a 60- or 10-cm culture dish.
In this case, cells can be detached mechanically by pipetting
them up and down, rather than tapping the flask.
6. After 2 days, cells should be 80% confluent, and they can be
split again. Cells should be split a minimum of two times before
seeding them in an MMI live chamber dish to ensure cell
viability. Once the cells have been split two times, they can be
30
Ana Gordon and Karine Gousset