Principle:
Mononuclear phagocyte progenitor cells derived from femoral and tibial bone marrow are propagated in the presence of M-CSF. This macrophage growth factor is secreted by L929 cells and is used in the form of L929 cell conditioned medium. The progenitor cells proliferate and differentiate through monoblast, promonocyte and monocyte stages before maturing to macrophages. At this time the cells become firmly adherent to the culture vessel.

Materials:
1. Dulbecco's minimal essential medium (DMEM) from Invitrogen Canada#11960-069

2. Heat-inactivated fetal bovine serum (Invitrogen Canada). This should be a tested for support of macrophage growth and needs to be of low endotoxin content (<.05ng/ml).

3. Stock penicillin/streptomycin /glutamine(10,000 units/ml , 10,000 µg/ml and 29.2mg ) Invitrogen, Canada #10378-016

Preparation of Bone Marrow Growth Medium:
1. Add 5 ml of stock penicillin/streptomycin/ glutamine to a 500 ml bottle of DMEM. Remove 100ml and save in a clean 500 ml bottle.

2. Add 50 ml of heat-inactivated fetal calf serum and 50 ml of L-cell conditioned medium, ( 25ml week1 and 25ml week2 ) and mix well.

Isolation of Mouse Bone Marrow Cells
1. Sacrifice mouse using CO2 and saturate mouse with 70% alcohol.

2. Clip the skin mid-back and remove the skin from the lower part of the body.

3. Remove tissue from legs with scissors and dissect away from body.

4. Clean remaining tissue from the femora and tibia and separate at knee joint. It is important to make sure that all the tissue is removed from the bones since cells associated with this can contaminate the marrow preparation and potentially overgrow the macrophages.

5. Cut off each end of bone.

6. Using a 25g needle and a 12cc syringe filled with bone marrow medium, expel the bone marrow from both ends of the bone with a jet of medium directed into a 50ml screw top Falcon tube.

7. Centrifuge, 1000RPM 10 minutes. Resuspend cells in 10ml bone marrow Medium. Using a 18g needle attached to a 12cc syringe, gently aspirate and expell the marrow until the cell aggregates have been broken up. Bring sample volume to 40ml with bone marrow medium.

8. Count cells

Culture of Bone Marrow Cells:
1. Adjust the cell suspension to a concentration of 11.0 x 106 cells/ml at dispense into culture dishes as follows:

6 well plates 4 ml/well
12 well plates 2 mls/well
24 well plates 1 ml/well
100 mm dishes 12 ml/dish

N.B. Regular tissue culture plastic plates should be used for adherent cells e.g. for RNA work, and bacteriological grade plastic dishes can be used for later work with cell suspensions. The cells can be removed from bacteriologic dishes be gently scraping with a rubber policemen or by jetting medium over the cell monolayers.

2. Incubate cells 5 to 7 days at 37° under a 10% (v/v) CO2. Feed the cells by adding 6 well plates 2 ml/well
12 well plates 1mls/well
24 well plates 0.5 ml/wel
100 mm dishes 5 ml

on day 5 and completely change the growth medium on day 6