freezing 3T3L1 cells and making lysates of four cell types

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BIOE 202 Lab 6
Feb 26
freezing 3T3-L1 cells and making lysates of four
cell types
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Pre-Lab
Review
Reasons for freezing cells include
Protection of valuable resource Cell lines in
continuous culture are prone to variation Finite
cell lines may become senescent Continuous cell
lines may prone to genetic instability Contaminati
on by microorganisms
Cross contamination by other cell lines Incubator
failure Saving time and materials maintaining
lines not in immediate use Need for distribution
to others
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Pre-Lab
Review
Cells should not be frozen quickly (1C/min is
preferable) Cells are frozen in a higher
concentration of serum and in DMSO (dimethyl
sulfoxide) The most important thing to remember
when using the centrifuge is that tubes must be
balanced The most important thing to remember
when producing lysate is to keep the samples cold
to impair enzyme function
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Pre-Lab
Review
Lysosomes release destructive enzymes during
lysate production PC-12 is derived from the
adrenal gland and takes on a neuronal phenotype
when cultured on collagen HeLa was the first
aneuploid line maintained in continuous
culture Leibovitz L-15 is best suited for non-CO2
equilibrated environments RPMI is suitable for
suspension cultures
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Freezing
Procedure
Trypsinize, place suspension in conical tube,
then count as usual Calculate the necessary
amount of freezing media (pg 61 of the lab
manual) Centrifuge (remember to balance) Label
your cryovials during the wait
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Freezing
Procedure
Remove your tube from the centrifuge and make
sure theres a white pellet Aspirate the media
without disturbing the pellet place supernatant
into waste bottle Add 2ml freezing media and mix
by pipette until the pellet is completely broken
up (you shouldnt see any flakes) Add the rest of
your freezing media
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Freezing
Procedure
106 cells in each vial (capacity 1ml) You may
freeze up to 2 vials bleach the rest of your
cells Place your vials on the rack in the
refrigerator
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Lysate
Procedure
Aspirate media from wells Rinse 2x with cold
serum-free media Rinse 2x with PBS Add SDS-PAGE
sample buffer Swirl buffer and use scraper to
lift cells off Place in sample tubes CHANGE TIPS
AND SCRAPERS BETWEEN SAMPLES
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Lysate
Procedure
Sonicate each tube Spin in microfuge Vortex Spin
in minifuge Aliquot 50µl samples into 0.5 ml
tubes then freeze Note the difference between
amount of buffer used for L1s and E63s
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Reminders
After today you should not have any cells left in
the incubators No return day enjoy your day
off Please e-mail your last post-lab exercise to
me