DC generation with Bone Marrow Cells

4 posts / 0 new
Last post
coltifer
coltifer's picture
DC generation with Bone Marrow Cells

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.

Culture
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

marcus muench
marcus muench's picture
Cell death suggests that

Cell death suggests that either the cells were overgrown or had reached the end of their natural lifespan. My guess is that they were overgrown, do you than that is a possibility? Did the medium turn yellow? FBS can also be a variable factor. I usually avoid FBS and go serum-free, but if needed I select FBS batches that don't cause much growth of hematopoietic cells in the absence of cytokines, but gives a high growth response when cytokines are added. I also heat-inactivate the FBS.

Minor points (I don't think these are a major issue for you):

1) It is possible to harvest BM without removing all the tissue. I simply hold the whole leg with forceps, cut the end off the femur, insert a 26 or 27 gauge needle that has been bent to a V-shape and then flush 2-4 ml of PBS through the femur while holding over a 50ml tube. The BM is effectively flushed from the femur with much less work. If you want you can then cut just below the white patch of ligaments at the knee and repeat the procedure to flush the tibia. You can also flush the humerus to maximize your marrow harvest from each mouse.

2) I'm not sure what the thinking is behind using DMEM, but I always use IMDM when growing hematopoietic cells. I really don't know how much difference it makes in this case, but I thought I'd mention it.

coltifer
coltifer's picture
They don't appear overgrown.

They don't appear overgrown. They're just aren't that many cells by the end of the protocol time and most of them look dead. The medium is a normal pink color. I might try not using FBS, even though ours is heat inactivated. Serum free conditions are sufficient for the DCs to grow?

marcus muench
marcus muench's picture
I grow human DC in serum-free

I grow human DC in serum-free, not mouse. When I've grown mouse myeloid colonies (myeloid, not necessarily DC) I've found the serum-free formulation is still improved with some added serum (5-10%, rather than 20%).

Can you try the same protocol with IMDM 20%FBS - that is an easier change than going down the serum-free path? Although I haven't tried that particular protocol, the length of time and cytokine conditions etc all sound about right, so I'm not sure what could be going wrong for you. Have you tested the cytokines and FBS in colony assays or some other growth assay on mouse BM? GM-CSF should really support a decent amount of growth from mouse BM.