CFC assays

CFC assays are performed in:

• 1.2% methylcellulose
• 30% FBS
• 57.2 uM b-mercaptoethanol
• 2 mM Glutamine
• 0.5 mM hemin (Sigma)

We use prefab mix from StemCells without cytokines.

We add the following cytokines:

• 20 ng/ml Interleukin-3 (IL3), 20 ng/ml IL-6, 20 ng/ml G-CSF, 20 ng/ml KL and 6 U/ml EPO.

Alternatively: use StemCell CFCmix GM (contains IL-3 and GM) and add EPO and KL (and evt IL-6, G and hemin).

CFU-E assays are performed as CFC assays in the presence or absence of EPO only.

CFC mixes including cytokines are aliquoted in 2ml/tube (-20 retrolab), add cells, split into two pertridishes.

Fresh/transduced day 5 CB: 1000 cells/plate

Fresh BM/PBSC: 2000 cells/plate

Expanded cells from MS5 coculture/liq culture: 10.000 cells/plate