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Salmon Lab Protocols: Tissue Culture

Mixing F-12 (HAM) Media
1) Start by rinsing a graduated cylinder three times ddH2O.
2) Then fill the cylinder to 600-ml with ddH2O.

3) Add one bottle of F-12 (HAMS) (Sigma #N-4388).
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) Add 5 mLs Sodium Pyruvate/Liter of media

7) Next pH the solution to 7.15 with 1N NaOH.
8) Bring the volume up to 890-ml with ddH2O.
9) Split the solution into two portions, each being 445-ml.
10) 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.
11) 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

  1. Set up your new flasks and coverslips prior to doing anything to the cells ready to be split.
  2. Using the vacuum tube, remove the old media.
  3. Rinse the cells with 6-7 ml of Sterile PBS and allow to sit for a few seconds.
  4. Using the vacuum, remove the PBS.
  5. 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.
  6. Using the vacuum, remove the trypsin.
  7. 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.
  8. Using the vacuum, remove the trypsin.
  9. Place back in the incubator and allow to sit until cells are floating. Time will vary depending on cell line ~about 4 minutes.
  10. During this period, ready the new flasks and dishesPut 7-ml media in the new flasks and 3 ml in the dishesLeave the caps loose.
  11. 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.
  12. 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.
  13. 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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