*Please note:
lately (2005 - present), we normally culture PtK1 cells in F-12 media,
but they also grow in other types of media as described below.
Mixing
F-12 (HAM) Media*
(*Sigma stopped making
the powdered media we used in the past - Sigma #N-4388; so we now use
powdered media from Gibco #21700-075 or liquid media from Gibco #11765-054.
Neither of these have Hepes buffering)
1) Start by rinsing a graduated cylinder three times ddH2O.
2) Then fill the cylinder to 600-ml with ddH2O.
3) Add one packet of F-12 (HAMS) (Gibco #21700-075).
4) Stir this until all of the powder is dissolved. (Note: Do not heat
to quicken the process.)
5) Once all of the media is dissolved, add 1.176-g NaHCO3
(Sodium Bicarbonate) and stir until dissolved.
6) Next pH the solution to 7.15 with 1N NaOH.
7) Bring the volume up to 890-ml with ddH2O.
8) Split the solution into two portions, each being 445-ml.
9) Sterile filter each one seperately. (This gives you two incomplete
batches that can be stored until needed). Check the pH, it should be
at 7.2- adjust accordingly.
10) When the media is needed, add 50-ml of FBS (Fetal Bovine Serum)
and 5-ml of antibiotics to one of the portions. This completes the media
for use.
MEM
for GFP histone PtK1s
1) rinse cylinder well with ddH2O
2) add 600 mls ddH2O
3) add one bottle MEM (Sigma #M-0643)
4) add 10uM Hepes
5) pH to 7.2 with 1N NaOH
6) bring up to 900 mls with ddH2O-test
pH
7) add 5 mLs of NEAA (non-essential amino acids, keep this sterile)
8) 450 mls media per sterile filter container
9) re-pH small sample poured into Falcon tube to assure filtering did
not change pH of media (this sometimes happens)
10) add fungizone, antibiotics (pen and strep) and FBS to one label
complete MEM
11) just add media to other and label incomplete MEM
12) add G-418 to 60ug/ml for GFP histone selection. (You may want to
add this individually to small volumes of media for use depending on
the amount of media you'll go through per unit time. The G-418 isn't
very stable to repeated heating/cooling.)
MEM
for PtK1s
1) rinse cylinder well with ddH2O
2) add 600 mls ddH2O
3) add one bottle MEM (Sigma #M-0643)
4) add 0.85 g Sodium bicarbonate
5) add 5 mls Sodium Pyruvate (keep this sterile)
6) pH to 7.2 with 1N NaOH
7) bring up to 900 mls with ddH2O-test
pH
8) 450 mls media per sterile filter container
9) re-pH small sample poured into Falcon tube to assure filtering did
not change pH of media (this sometimes happens)
10) add fungizone, antibiotics (pen and strep) and FBS to one label
complete MEM
11) just add media to other and label incomplete MEM
DMEM for PtK1s
1) rinse cylinder well with ddH2O
2) add 600 mls ddH2O
3) add 1 bottle DMEM
4) add 3.7g Sodium bicarbonate
5) pH to 7.2 with 1N HCl
6) bring up to 900 mls with ddH2O-test
pH
7) 450 mls media per sterile filter container
8) re-pH small sample poured into Falcon tube to assure filtering did
not change pH of media (this sometimes happens)
9) add fungizone, antibiotics (pen and strep) and FBS to one label complete
DMEM
10) just add media to other and label incomplete DMEM
L-15 for PtK1s
1) rinse cylinder well
with ddH2O
2) add 600 mls ddH2O
3) add 1 bottle L-15 (Sigma #L-4386)
4) add 7mM Hepes (1.822g for 1L)
5) pH to 7.2 with NaOH
6) bring up to 900 mls with ddH2O-test
pH
7) 450 mls media per sterile filter container
8 re-pH small sample poured into Falcon tube to assure filtering did
not change pH of media (this sometimes happens)
9) add fungizone, antibiotics (pen and strep) and FBS to one label complete
L-15
10) just add media to other and label incomplete L-15
Phenol
Red-Free L-15 with High Glucose for filming PtK1s
1) Buy Gibco
Leibovitz's L-15 medium with L-glutamine, without phenol red, cat.#
21083-027
2) Add 4.5 g/L (2.25 g/bottle) glucose (Sigma #G-6152).
3) Add fungizone, antibiotics (pen and strep) and FBS.
Splitting Cells
- Set up your new flasks and
coverslips prior to doing anything to the cells ready to be split.
- Using the vacuum tube, remove
the old media.
- Rinse the cells with
6-7 ml of Sterile PBS and allow to sit for a few seconds.
- Using the vacuum, remove
the PBS.
- Put 1.0 ml of trypsin
in the flask, move flask around and hit it on the bottem against the
table (hood) to dislodge floating cells.
- Using the vacuum, remove
the trypsin.
- Put 1.0 ml of trypsin
in the flask, move flask around and hit it on the bottem against the
table (hood) to dislodge floating cells.
- Using the vacuum, remove
the trypsin.
- Place back in the
incubator and allow to sit until cells are floating. Time
will vary depending on cell line ~about 4 minutes.
- During this period, ready
the new flasks and dishes. Put 7-ml
media in the new flasks and 3 ml in the dishes. Leave
the caps loose.
- When you remove your
cells from the incubator, bang the flask on a
counter or against your hand to separate the cells from one another.
Observe them to make sure they are free floating.
- To remove the cells, draw
5 ml of fresh media and wash it over the bottom
of the flask. Draw up the same 5-ml and repeat two
or three times. Next keeping the tip of the pipet in the
fluid, draw up most of the media and then flush
it back out into the media itself. Then draw it
all up and wash the bottom one final time.
- Draw up all of the
liquid and put the desired amount in your new containers.
For our lab, using PtK1 cells, the standard distribution
is in a 1 to 3 or 4 ratio for a new flask and 5-9 drops for a coverslip
dish. Again this ratio will vary with your cell line.
Use of ciprofloxacin-treated
media:
Ciprofloxacin is
an effective antimycotic agent that can be purchased from most hospital
pharmaceutical stock rooms. It is often found under the name Cipro
I.V., produced by Bayer Corporation. It does a quite a number on fungi
and mycobacteria, leaving tissue culture cells free of their awful
influence. Two hundred-milligram Cipro is diluted from 10 mg/mL stock
to 10 uL/mL (i.e. 1:1000) in complete F-12 medium (including
both FBS and the usual pen/strep/amp mixture). Before introduction
of drug-treated medium to a flask or petri dish, old media should
be sucked off, and cells should be rinsed with sterile PBS. Cells
can then be fed or split as usual, using the ciprofloxacin medium.
A cell line should be subjected to this treatment for one week, starting
from the first day of introduction of drug-treated medium by feeding
or splitting. It is important that flasks be labeled as to when the
first day of drug treatment was, so that the treatment is not carried
on for too long a period of time. When the prescribed period of time
has elapsed, cells should be examined for the continued presence of
foreign organisms; this is done most effectively by staining cells
with DAPI (a DNA-intercalating dye), as bugs will appear as little
spots external to your cells' nuclei. Treatment should be resumed
for another 3-4 days if you do see bugs during the initial check-up,
and discontinued immediately if no foreign organisms are detected.
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