General Protocol for LCL culture

Author: Orsolya Symmons

Date: 2015-04-29

A good resource for general questions is the Coriell website


Recommended media (warm in a 37ºC water bath for 20 minutes before every use!):

            RPMI 1640 (1X)                              1L bottle

            15% Fetal Bovine Serum (FBS)        176 mL

            2mM L-glutamine                             10 mL

            100U Penicillin Streptomycin           10 mL

Recommended culture conditions:

            ~ T25 tissue culture flask with 10-20 mL of medium

            ~ Store flasks with growing cells in an upright position

            ~ Keep incubator at 37ºC under 5% carbon dioxide

Waking up frozen cells:

  1. Warm the media in a 37ºC water bath.
  2. Label 15mL tubes and add 6mL of warm media to each tube.
  3. Remove the frozen cells from LN2 and circulate quickly in a 37ºC water bath until just thawed from ice state. Wipe down tubes with 70ºC EtOH and place in cell culture hood.
  4. Pipet mix cells once, and transfer entire volume to 6mL of aliquoted media. Gently pipet mix twice.
  5. Spin down the cells for 6 minutes at 710prm RT.
  6. Aspirate all the media off of the pellet, add 10mL fresh warm media to the tube and carefully resuspend the pellet.
  7. Transfer 10mL to the final labeled flask.

Maintaining and splitting cells:

  • Usually appropriate to split the cells once every two days following these conditions:
    • Never let the cells reach a concentration of 1 million cells/mL.
    • Split to a final concentration of approx. 300K cells/mL, never splitting to less than 200K cells/mL.
    • Never let the media get very yellow, since this means that there is high acidity in the culture. Try to maintain them at a pinkish color.

To double the volume of cells (splitting the concentration in half):

  1. Add 10 mL of warm media to the flask and pipet mix about three times with the intention of breaking up any cell clumps at the bottom of the flask. To do this, place the serological pipet tip against the bottom of the flask when pipetting out to break the clumps with pressure.
  2. Transfer 10 mL of mixed cells to a new labeled flask so both flasks now have 10mL of cells.

To maintain the same volume of cells (splitting the concentration in half):

  1. Swirl the flask to mix all the settled cells back into the media.
  2. Aspirate off 5 mL of media from the cells, while moving around the Pasteur pipet to aspirate evenly from all areas of the flask.
  3. Add 5 mL of warm media and pipet mix three times.

Pelleting cells for further use (RNA/DNA/protein extraction):

  1. Swirl the flask(s) of cells to resuspend all settled cells into media.
  2. Transfer a volume with the desired number of cells into an appropriate tube (15mL or 50mL).
  3. Spin down the cells for 7 minutes at 700rpm RT.
  4. Aspirate off all media.
  5. Wash 1x or 2x with 5mL PBS:
    1. Resuspend the pelleted cells in 5mL PBS.
    2. Spin for 7 minutes at 700rpm RT.
    3. Aspirate off all PBS and repeat.
  6. After final PBS wash, aspirate off all PBS and freeze at -80ºC until further use.

Freezing cells:

  • Whatever media the cells are being grown in is the same media used to freeze them.
  • Freezing media:
    • 70% Media 14 mL
    • 20% FBS 4 mL
    • 10% DMSO2 mL
    • Before starting, it is best to have all tubes labeled. All labels should include name of cell line, date, your initials, and (if possible) the cell passage.
  1. Place cell suspension in a 15mL tube and spin at 700rpm for 5 min RT.
  2. Aspirate off all media without disturbing the cell pellet.
  3. Add 1mL of freezing media to all cell pellets.
  4. Gently resuspend cells and place each cell pellet suspension into a 2mL cryovial tube.
  5. Place cryovials into a Mr. Frosty freezing container and place in -80ºC for a minimum of 4 hours. [They can be left in -80ºC for up to a few months.]
  6. Transfer to a box in a LN2 tank for long-term storage.