Making conditioned medium for monoclonal LCL production

Author: Orsolya Symmons

Date: 2015-04-29


  1. Seed approx 20-25 flasks with 20ml of ~300.000 cells/ml.

Underlying logic: no more than 20ml should be cultured in a 25ml flask, and this way the total volume of cultured medium will approx fill a stericup+top filtering device.

  1. After 24-28h, pool medium into 50ml Falcon tubes, spin @1000rpm for 5'.
  2. Remove medium, transfer to 0.2µ filtering device.
  3. Filter.
  4. Aliquot into 10ml batches and freeze @ -20°C. Should be stable for multiple months.

            Use conditioned medium at a ratio of <40% relative to “normal” LCL medium.


Making monoclonal cell lines from LCL cell lines


1. Collect cells, spin, resuspend and count

2. Spin cells and resuspend in a mix of conditioned/normal (I used ratio of 2:3) medium at a concentration of 5-7 cells/ml

3. Aliquot 100µl of cell suspension into the wells of a 96-well plate. Making monoclonals has very low efficiency, so you will need about 5-10plates/cell line.

4. Wait for approx 4 weeks. Some of the medium will evaporate, so you might need to top up with approx 50µl of medium mix after approx 2 weeks. You can start looking for yellow wells after about 3 weeks.

5. If you find wells with growth, resuspend and seed them into 48-, 12-, 6-well plates.

Useful info: here