I am looking for some different ways to make DCs differentiate.
Hi Gabriel Cordeiro ,
I don't know whether this would help but I am using Flt3 ligand to to generate BMDCs. It is very straightforward:
- Take out the femur and tibia (x2 per each per mouse).
- Spray the bones quickly with ethanol and then rinse them with PBS.
- Prepare cell culture media/flt3 ligand mix, 20 mL/mouse in PD100 (18 mL media + 2 mL supernatant; I think this might need optimisation depending on the supernatant)
- Cut the end of the bones and flush the content of the bone with a syringe/appropriate needle combo into the cell culture media/flt3 ligand mix (I can give you the recipe for the media if needed).
- Incubate the media+ligand+cell mix for 8-9 days for differentiation.
- If you want to activate the BMDCs, use LPS (100 ng/mL) for 16 hours, then wash it off.
Cheers
