- Spin down the media at 1500gfor 10 min to remove any
detached cells. - Pool the medium collected from all flasks and filter-sterilize
media using a 0.22μm, bottle-top filter. - Aliquot and store L929-conditioned media at 80 C.
BMDM Isolation- Anesthetize and euthanize mouse by overdose of ketamine
(150 mg/kg) and xylazine (10 mg/kg) or equivalent locally
approved protocol. - Spray the mouse with 70% ethanol.
- Expose the musculature on each leg by removing the skin
manually or with scissors (seeNote 2). - Remove each exposed leg by gently cutting around the head of
the femur to detach ligaments and musculature, while being
careful not to cut or crack the femur (seeNote 3). - Surgically remove all muscles from each leg (seeNote 4), and
then sever tendons between the tibia and femur (discard
fibula). - When the bones are devoid of muscle, cut the ends off each of
the bones, and then place the femur and tibia from one leg into
a sterile 0.5 mL tube (which has had a hole punctured by a
sterile 18-gauge needle at the very bottom). - Place the 0.5 mL microfuge tube containing bones in a sterile
1.5 mL tube, and add 100μL of BMDM medium to the
0.5 mL tube. - Centrifuge the tubes on a benchtop microcentrifuge at
3000 gfor 5 min. - Dispose of 0.5 mL tube containing bones (seeNote 5).
- Add 900μL of BMDM medium to the 1.5 mL microfuge tube
containing the pelleted bone marrow and gently resuspend (see
Note 6). - Pipette the resuspended bone marrow through a 40μm cell
strainer fixed on a sterile 50 mL conical tube. - Pass an additional 25 mL of BMDM medium through the
40 μm cell strainer. - Transfer the bone marrow media into a T175 flask containing
60 mL of BMDM medium (making the final volume 85 mL). - Incubate cells in a humidified 5% CO 2 incubator at 37C for
4 h so that differentiated cells adhere. - Add 15 mL of L929-conditioned medium to the T175 flask
and mix thoroughly (seeNote 7).
484 Nicholas D. LeBlond and Morgan D. Fullerton