Eosinophil Liquid Cultures from LDBM Cells

Low Density Bone Marrow Harvest

  1. Remove hind legs of mouse at hip bone?), scrap away muscle, and place in PBS
  2. Crush bones in a mortar and pestle under hood
  3. Filter through a 70um cell strainer Into a conical tube
  4. Wash bones one more time by adding 10 mls of 1X PBS, filter , and add to previously filtered cells.
  5. Spin at 1200 rpm for 5minutes
  6. Remove super, add 10 ml RBC lysis buffer
  7. Incubate two minutes at 37C with swirling
  8. Add 20 ml of 1X PBS, or 1X HBSS buffer
  9. Spin at 1200 rpm for 5minutes
  10. Remove Super
  11. Add 3 ml of 1X HBSS buffer to cells/per two mice with which you started

(so, if you started with 4 mice, add 6 ml)

  1. Add 3 ml of histopaque 1083 to a 15 ml conical tube
  2. Layer 3 ml of cell in HBSS over the histopaque, very slowly, so that you have two clean layers
  3. Spin tubes at 1700rpm for 30 min
  4. LDBM cells will remain at interface
  5. Aspirate off the top layer with vaccum in tissue culture hood , down to cells
  6. Pipette off the cells into new tube
  7. Add 2X the volume of cells with IMDM media and mix
  8. Spin the cells at 1200 rpm for 5 minutes
  9. Remove super, resuspend cells in media, count

Generally a 4-week old mouse will give approximately 10 X 106 LDBM cells

7-week old mouse will give approximately 5 X 106 LDBM cells

Eosinophil Culture

  1. Plate LDBM cells at 1 X 106cells/ml

2.Change media every two days by carefully removing the media without disturbing the cells

3.To day 0, and day 2 media add SCF, FLT3 at 100 ng/ml each

4.All media changes from Day 4 onward should contain 10 ng/ml IL5

Eos Culture Media

IMDM + Glutamax (GIBCO 31980)

Pen/Strep 5-ml (Gibco 15140)

FBS 10% (use Hyclone one, which is better for this purpose)

L-Glutamine 5-ml of a 200mM (Gibco 25030)

B-ME final concentration 55um or 1X (Gibco 21985-023)