Culture of Mouse Bone Marrow Cells

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Mouse bone marrow cells are truly multipurpose. Here are instructions for creating three difference cell types from mouse bone marrow.

Materials for Macrophages

  • DMEM with 10% FBS and Hypoxanthine-Thymidine Added (HT supplement available from Invitrogen)
  • Mouse Bone Marrow Cells (catalog # 1060)
  • M-CSF (also called CSF-1) (can use human or mouse recombinant)

Procedure for Macrophages

  1. Thaw bone marrow cells following standard procedure and wash once.
  2. Adjust cell number to 5 x 105/mL in medium.
  3. Add M-CSF to the medium at 20 ng/mL.
  4. Put cells into desired culture vessel (flask, multiwall plate).
  5. Incubate at 37°C, 5% CO2 for 3–4 days.
  6. After 3–4 days incubation, there should be colonies of macrophages present. Continue to change the medium every 3–4 days until cells fill the available surface. Cells will begin to pile up after this point.

Materials for Dendritic Cells

  • Recombinant Mouse GM-CSF
  • Bacteriologic Grade Petri Dishes

Procedure for Dendritic Cells

  1. Thaw bone marrow cells following standard procedure and wash once.
  2. Adjust cell number to 3 x 105/mL in medium.
  3. Add GM-CSF to the medium at 20 ng/mL.
  4. Put 10 mL of cell into each dish and incubate at 37°C, 5% CO2 for 3–4 days.
  5. Add 10 mL of fresh medium containing 20 ng/mL GM-CSF to each dish, then return dishes to incubator.
  6. On day 9, collect non-adherent cells from the culture. These should be all dendritic cells with some expression of CD86. The adherent cells are mostly macrophages.

Materials for Mast Cells

  • Recombinant Mouse SCF
  • Recombinant Mouse IL-3

Procedure for Mast Cells

  1. Thaw bone marrow cells following standard procedure and wash once.
  2. Adjust cell number to 5 x 105/mL in medium.
  3. Add IL-3 to the medium at 100 U/mL and SCF at 50 ng/mL.
  4. Culture cells in a standard T75 flask at 37°C, 5% CO2 for 3–4 days.
  5. After 3–4 days, collect non-adherent cells into a centrifuge tube and centrifuge at 200xg to pellet the cells. There will be adherent macrophages left behind in the flask.
  6. Decant the medium and resuspend the cell pellet in fresh medium containing IL-3 and SCF.
  7. Repeat steps 5 and 6 two more times. At this stage, most of the cells will be mast cells as shown by staining for Fc epsilon receptor a c-kit (CD117).