L 929 cells are maintained in T-75 cm2 filter cap flasks in DMEM( Invitogen, Canada #11960-069) + 1% Penicillin Streptomycin / L-glutamine (Invitogen, Canada #10378-016) + 10% heat inactivated Fetal Bovine Serum (56°C,30 min.) and 7.5% CO2.

The Fetal Bovine Serum is the same as used for cultivation of the Bone Marrow Derived Macrophages. Serum is tested for use as outlined in serum testing protocol.

Cells are split once every week by removing media from on T-75 flask, adding 10ml of warm Calcium and Magnesium free Hanks Buffered Saline. Wash cell surface by tipping back and forth about ten times,remove wash solution , add 1.0 ml of Trypsin- EDTA (#25300-054) Invitogen, Canada. Place back in incubator for not more than 5 min. at 37°C. Add 10 ml culture medium, and with a 10ml pipette rinse cells from flask surface, as well as up and down to break up the clumps of cells. Add 2 drops of this suspension to 20 ml of culture medium in each of (2) 75cm2 flasks. (10 ml of culture medium can be added to the trypsinized flask and kept as a backup if desired.) Incubate for 7days and repeat above to maintain culture. Maintain (2) flasks so that one can be used to split cells and the other to serve as a backup in case of contamination.

To produce LCCM, set up (8) T-150 800ml Triple Flasks with filter caps (Nunc #132913). Add 150 ml of culture medium to each flask. Count the cells in the trypsinized suspension ( To 800 µl culture media add 100µl Trypan Blue and 100µl cell suspension, mix and charge counting chamber. Count. Each 150cm2 surface requires 0.24 x 106 cells . For the triple flasks add 0.72 x 106 cells in 1.0 ml of culture medium. Use a 1ml pipette and add the 1ml in between the fins. Mix the cells and medium carefully in the flask. From the upright position of the triple flask, tip gently to the side( to maintain the equal level of media on each surface) away from the hole in the center of the bottom of the flask then lay flat. Stack four flasks high and incubate 7 days at 37°C, 7.5% CO2 . Several times during the week , tip the flasks upright and then lay flat again to equally distribute the cells on all the growing surfaces. At the end of the first week , pour off the media and filter 0.2µ . Add 150 ml fresh warm culture medium to each flask and incubate another 7 days, tipping as above. Filtered Week 1 LCCM is aliquoited (45 ml each) in 50 ml Falcon centrifuge tubes, and frozen at -20°C. At the end of the second week, pour off media and discard flasks and cells. Filter Week 2 LCCM 0.2µ. This will be difficult as there is a lot of cellular debris , requiring several filters. Aliquot into 50 ml tubes as above. LCCM is stable page 2 for about one year at -20° C. Test LCCM using a bioassay of Bone Marrow Derived Macrophage growth.

To test LCCM plate 11 x 106 mouse bone marrow cells in 24 ml Bone Marrow Medium ( DMEM + 1% PenStrep/ L-glut +10% heat inactivated serum + 10% of either Week 1 OR 10% Week 2 LCCM) in a 12 well Tissue Culture dish. Allow to grow for 5 days, without feeding at 37° C 10% CO2 . Count (3) wells for each week of LCCM. Growth should be about 1 x 106 Cells per well for Week 1 and about 1.8 x 106 cells per Well for week 2. It has been found that a 50/50 mix of Week 1 and Week 2 used at 10% in the BMM will provide the correct growth rate of the Bone Marrow Derived Macrophages, neither over grown nor too sparse. Cells should look healthy and not activated.