cytokine-driven liquid culture differentiation assays

Protocol for unilineage-restricted differentiation of human CD34 cells

 

 

medium: QBSF without serum, two weeks

start with 10.000 cells

 

Erythroid differentiation:

 

Epo (3 U/ml), IL-3 (0.01 U/ml), GM-CSF (0.001 ng/ml)

 

Granulocytic differentiation:

 

IL-3 (1 U/ml), GM-CSF (0.1 ng/ml), G-CSF (500 U/ml)

 

Megakaryocytic differentiation:

 

Tpo (100 ng/ml), IL-3 (0.01 u/ml), KL (10 ng/ml), IL-6 (10 ng/ml)

 

Monocytic differeniation:

 

40% FCS, IL-6 (1 ng/ml), FL (100 ng/ml), M-CSF (200 U/ml)

 

B-cell differentiation:

 

IL-7 (100 ng/ml), FL (10 ng/ml)

 

 

 

Protocol - liquid culture cytokine-driven differentiation of human CD34 cells

 

plate 50.000 transduced CD34+ cells in 4 ml Iscove’s modified Dulbecco’s medium (IMDM, Gibco-Life technologies) in the presence of 10% FBS, IL-3 (20 ng/ml), IL-6 (20 ng/ml), c-Kit ligand (KL), Flt-3 ligand (FL) and thrombopoietin (TPO) (20 ng/ml each)