Pelling:Protocols/Mammalian Cell Culture

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Contents

Reagents Needed

Thawing Cells and Beginning Culture

  1. Remove an appropriate cryovial from the LN2 tank and hold in the 37°C water bath. The O-ring and cap should be kept out of the water bath to prevent contamination. Once it is almost completely thawed (a small amount of ice should be observed in the tube) remove the vial from the water bath.
  2. Wipe the cryovial with EtOH and bring to the hood.
  3. Remove the cells with a sterile disposable plastic pipette and dispense into a 15ml tube which contains 5ml of complete medium, bringing your total to ~6ml. Resuspend the cells by mixing with the pipette.
  4. Pellet the cells by centrifugation at 1000 RPM for 3-5 minutes.
  5. While waiting for the centrifuge, prepare a T75 flask with 13 ml of medium. Label the flask with the Cell Type, Your Initials, Date and Passage # (use the P# on the cryovial).
  6. After centrifugation is done, use a 10ml pipette to remove the supernatant medium into the waste beaker leaving behind the pellet.
  7. A good technique is to tap the bottom of the tube gently against the hood surface several times to break up the pellet. This prevents cell clumping when the medium is added and the pellet should now act like a viscous liquid instead of a hard clump.
  8. Re-suspend the pellet in 2ml of media. Suspend several times until the pellet is completely mixed with the medium and no cell clumps are present.
  9. Dispense the re-suspended 2ml into the T75 (which already contains 13ml of media). Therefore your total volume is 15ml.
  10. Using a light microscope, the cells can be visualized as round balls. They will begin attaching and spreading in ~ 45-60min.
  11. Place in the incubator overnight and add change to a fresh 15mL media the next day.
  12. Split cells when they are ~70-80% confluent (can take 1-5 days depending on cell type). Media should be refreshed every other day if cells take > 2 days to reach confluence. NEVER let cells become over-confluent (>90%) unless you are doing so deliberately.

Changing the Media

  1. Warm complete DMEM in 37°C water bath for 15min.
  2. Remove old media from cells and replace with an equal volume of new media
  3. Place cells back into the tissue culture incubator.

Splitting Cells (Cell Passage) for T75 Flasks or 100mm Plates (Use Half Volumes for T25 or 60mm Plates)

Splitting occurs when “growing up” cells to make frozen stocks or when a “working stock” becomes ~70-80% confluent. A working stock is maintained in a 100mm plate and is your day to day stock of constantly growing cells which you use for experiments. Typically, working stocks are split in 1:10, 1:20 or 1:40 dilutions where a small amount (one tenth, one twentieth or one fortieth, respectively) of the cells are placed into a new 100mm plate, and the rest are either discarded or plated onto smaller plates/coverslips for experiments (staining, AFM, confocal etc). An optimal dilution is determined so that working stocks are typically split twice a week (Mondays and Thursdays).

  1. Thaw 50mL TE in 37°C water bath if you have not already done so. Otherwise, warm your 50mL aliquot of TE in the water bath.
  2. Remove all of media from the cell culture
  3. Add 10 mL of PBS to wash away the remaining media. All media should be removed because the TE will react with proteins in the media.
  4. Add 5mL of TE. Trypsin is a protease used to denature proteins including those attaching the cells to the substrate. TE is detrimental to cell function if left on for too long. EDTA: chelates Ca2+ ions in the solution which are involved in cell attachment.
  5. Place in the tissue culture incubator for 3-5 minutes at ~37°C. During this time, put the TE back in the fridge if no longer needed. After 3-5 minutes cells will be floating freely in solution and are visible to the naked eye.
  6. Remove the solution of cells from the flask or plate and place into a 15mL tube with 5mL of media. Any remaining TE will be neutralized by reacting with the protein in the media.
  7. Use the 10mL suspension of cells to rinse the flask or plate to dislodge any cells that may still be attached to the surfaces and transfer all liquid back to the 15mL tube.
  8. Centrifuge tube for 5 minutes to get a cell pellet at the bottom of the tube.
  9. Prepare the new flasks or plates with an appropriate amount of media in each. For “growing up” cells to make frozen stocks prepare 3 x T75 flasks with 12mL of media (this is a 1:3 split). For a basic split of a “working stock” of cells, prepare a 100mm plate with 10mL of media. Prepare 35mm or 60mm plates with 2mL or 3mL of media for experiments. 6-well plates take 2mL of media in each well.
  10. After centrifugation, remove the supernatant and expel into the waste beaker leaving behind the pellet.
  11. A good technique is to tap the bottom of the tube gently against the hood surface several times to break up the pellet. This prevents cell clumping when the medium is added and the pellet should now act like a viscous liquid instead of a hard clump.
  12. Re-suspend the pellet in 2mL of media. Suspend several times until the pellet is completely mixed with the medium and no cell clumps are present.
  13. Dispense the appropriate amount of cell suspension into the new flasks/plates that you setup in step #8 to achieve the desired split ratio (1:3, 1:10 etc.). For example if you want to do a 1:40 split then you take 50uL from your 2mL of cell suspension and add it to your pre-prepared flask or plate. Note: 2mL/40 = 0.05mL = 50uL.

Freezing Cells

  1. Freezing Media is FBS with 10% DMSO. To make freezing media, thaw a 20mL tube of FBS in a 37°C water bath. Remove and discard 2mL of FBS and replace with 2mL of DMSO. Mix gently by inverting and DO NOT produce a lot of foam.
  2. Cells to be frozen should be grown in T75 flasks. The following protocol is based on ONE T75 flask. Volumes should be scaled up proportionately.
  3. Trypsinize cells as described above and centrifuge to form a pellet. Remove supernatant.
  4. After centrifugation add 5mL of freezing media and resuspend cells VERY GENTLY. FBS will foam and bubble if you are too vigorous during mixing and this will be a problem later.
  5. After resuspending cells, count them. A confluent T75 flask typically yields ~5x106 cells total (±20% or so).
  6. Set up 5 cryovials (1mL per cryovial) and label with Cell Type, P#, Date and Initials.
  7. Using a syringe, draw up the 5mL of cell suspension. Using a syringe helps to get rid of bubbles.
  8. Dispense 1mL of cell suspension into each cryovial and cap tightly.
  9. Place the cryovials into the “Mr. Frosty” freezing container (ensure there is the appropriate amount of isopropanol in it).
  10. Place “Mr. Frosty” into the -80°C freezer overnight.
  11. The next day place the cryovials into your rack in the LN2 tank.
  12. Record where the cells are in the rack AND the rack position in the tank.
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