I'm having trouble getting the following protocol to produce the numbers it should. Instead I get a lot of cell death.
Any advice? It's the Lutz method as described in his paper.
V Lutz Method JIM 223 (1999) 77-92
-Remove femurs and tibiae from mice and purify from the surrounding tissue.
-Cut both ends of bone with scissors and flush the marrow with PBS using a .45mm (25G?) diameter needle. Disintegrate clusters by vigorous pipetting.
-After one wash in PBS, 1-1.5 x 107 leukocytes should be obtained per femur or tibia.
Day 0: 2 x 106 leukocytes are seeded in 10ml DMEM + 10% FBS containing 200U/ml of GM-CSF per 100mm dish.
Day 3: Another 10ml DMEM + 10% FBS containing 200U/ml of GM-CSF is added to the plates.
Day 6: Half of the culture supernatant is collected, centrifuged, and the cell pellet resuspended in 10ml fresh DMEM + 10% FBS + 200U/ml GM-CSF.
Day 8: Half of the culture supernatant is collected, centrifuged, and the cell pellet resuspended in 10ml fresh DMEM + 10% FBS + 100ng GM-CSF.
Day 10: Cells can be used already or culture can be continued to further reduce granulocyte contamination.
Plates fed as on Day 6 and 8, but fresh 10ml of medium contains only 30-100U/ml of GM-CSF.
For complete maturation:
-Collect non-adherent cells by gentle pipetting.
-Centrifuge at 300g for 5min at RT.
-Resuspend in 10ml media in a fresh dish containing 100U/ml GM-CSF and LPS at 1ug/ml.
-Culture cells for 1 or 2 more days.
-should yield 1-3x108 BM-DCs per mouse after 10-12 days