L-929 cell protocol

 

Growth  (L-929 cells require DMEM/F-12 with 10% FBS. As always, maintain a
sterile environment when working with cell cultures)

 

1)   L-929 cell stock can be found in liquid nitrogen container

2)   Thaw cells quickly and resuspend in 5mL of medium 

3)   Spin cells down, remove supernatant

4)   Resuspend cells  in 10mL medium and put into T25 flask. Incubate.

5)   When cells are confluent, split into 4 T75 flasks in 10mL of medium. Incubate

6)   When cells are confluent, collect all cells from the T75 flasks and split them
into 10 T175 in 50mL of medium. Incubate.

 

Medium collection (Medium is to be collected after 7 days of adding fresh medium
to cells)

 

1)   Collect all 50mL of conditioned medium from each flask.

2)   Replace conditioned medium with 50mL of fresh medium to each flask. Incubate
flasks.

3)   Collected medium can be stored in freezer.

 

*Conditioned medium will be unfiltered. Filtration is required before use. Steriflip filters
  are found in TC room.