Eosinophil Liquid Cultures from LDBM Cells
Low Density Bone Marrow Harvest
1.
2.
3.
4.
Remove hind legs of mouse at hip bone?), scrap away muscle, and place in PBS
Crush bones in a mortar and pestle under hood
Filter through a 70um cell strainer Into a conical tube
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)
12. Add 3 ml of histopaque 1083 to a 15 ml conical tube
13. Layer 3 ml of cell in HBSS over the histopaque, very slowly, so that you have two
clean layers
14. Spin tubes at 1700rpm for 30 min
15. LDBM cells will remain at interface
16. Aspirate off the top layer with vaccum in tissue culture hood , down to cells
17. Pipette off the cells into new tube
18. Add 2X the volume of cells with IMDM media and mix
19. Spin the cells at 1200 rpm for 5 minutes
20. 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 106 cells/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)