Flint Group Protocols
(9) Transferring cell lines to larger TC flasks.
Neuro2A cell line.
Neuro2A cells defrosted from liquid nitrogen storage grew well after 4 days in the
37oC incubator. Examined the cells down the microscope and found there was ~7080% confluence, which is ideal. They now needed to be transferred to a larger,
250ML flask.
1) Safety cabinet: Remember to BOOK IT for a specific time. Also, when
booking, give yourself plenty of time to complete the job!
2) Must put MEME (growth medium for Neuro2A cells) in water bath half an
hour beforehand along with PBS to prewarm solutions.
3) Take off the front covering of the cabinet and press the first 3 buttons on the
left to switch on the airflow, mute the alarm and activate the lights. Wait for
the correct air flow to be established in the cabinet. Then spray down surfaces
with Virkon, followed by ethanol.
REMEMBER: Any items entering the cabinet must be sprayed and wiped
down with ETHANOL first.
4) Must dislodge the Neuro2A cells from the TC flask. Pipette the MEME
medium up and down over the bottom surface of the flask a few times, fairly
vigorously. Transfer to a 50ML Falcon tube (previously cleaned with
ethanol!). Then do the same with ~3ML of PBS to remove as many remaining
cells as possible. Transfer to the same Falcon tube.
5) Spin in the centrifuge at 900RPM for 5 minutes. Cells will pellet. Remember
to ethanol wipe Falcon tube before returning it to the cabinet.
6) Pipette off the solution from the Falcon tube without disturbing the cell pellet.
Discard in the DispoJar with Virkon.
7) Then add 10ML of fresh MEME to the Falcon tube and pipette up and down a
few times to resuspend cells.
8) Transfer the resuspended cell suspension to a new 250ML TC flask, covering
its bottom surface.
9) Examine the original flask to ascertain whether or not most of the Neuro2A
cells have been transferred to the new flask. If not, repeat the process (starting
at step 4) using, perhaps, 3ML of MEME medium to resuspend.
10) Incubate in 37oC incubator.
Leave the cabinet clean and tidy. Apply Virkon, followed by ethanol again.
Switch off the cabinet and replace the front covering.
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Flint Group Protocols
An Alternative Protocol for Maintaining Neuro2A cell lines.
1) Pipette off old medium and dead cells into DispoJar with Virkon.
2) Add ~4ML of prewarmed PBS into each 250ML flask and apply scraper to
surface to remove attached Neuro2A cells.
3) Transfer this solution to fresh 50ML Falcon tubes.
4) Add another ~10ML of PBS into each of the flasks and pipette up and
down several times, in an effort to remove any remaining cells within each
flask. Don’t be too vigorous with the pipetting. Transfer to the Falcon
tubes.
5) Look at the flasks down the microscope to see to what extent the attached
cells have been removed. If there are still many cells remaining, then
repeat the washing with PBS.
6) Spin the Falcon tubes at 900RPM for 5 minutes.
7) Pipette off the PBS without disturbing the cell pellets and thoroughly
resuspend the pellets in 10ML of prewarmed MEME solution. Don’t be
too vigorous with the pipetting.
8) Transfer solutions to new 250ML flasks (appropriately labelled) for further
incubation.
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