P-EMBL-64 - P-EMBL-64
Human cord blood (CB) was collected from the umbilical cord after informed consent using guidelines approved by the Ethic Committee on the Use of Human Subjects at the University of Heidelberg. Mononuclear cells (MNC) were isolated after centrifugation on Ficoll-hypaque (Biochrom KG, Berlin, Germany). CD34+ cells were enriched with a monoclonal anti-CD34 antibody labeled with magnetic beads on an affinity column (Miltenyi Biotec, Bergisch-Gladbach, Germany). CD34+ enriched cells were incubated with anti-CD38-phycoerythrin (PE) (Becton Dickinson, San Jose, CA, [BD]) and anti-CD34-Allophycocyanin (APC)(BD). The cells were washed in PBS, 10% FCS and stained with propidium iodide (PI) to identify dead cells. CD34+/CD38- and CD34+/CD38+ populations were sorted using the automatic cell depositing unit on a FACS-Vantage-SE flow cytometry system. Cells positive for PI were excluded. About 10000 to 20000 CD34+/CD38- cells were isolated form one cord blood sample.
The murine fetal liver cell line AFT024 (a kind gift from I. R. Lemischka, Princeton University, Princeton) was maintained in DMEM (Gibco, Grand Island, N.Y. USA) supplemented with 20% FCS, 50 µM 2-Mercaptoethanol (Bio-Rad, Hercules, CA) as described before. Cells were grown to confluency in 24- or 96-well plates precoated with 0.1% gelatin (Specialty Media, Lavette, NJ) and maintained at 33C. AFT024 cells were then irradiated with 2000 rads using a irradiator 24 hours prior to using the feeder layer.
CD34+/CD38- cells were divided in two equal fractions and cultivated either on an AFT024 or without a feeder layer in 24-well plates. Both fractions were cultivated in the same media that consisted of RPMI supplemented with 20% FCS (R20), Penicillin 1000 U/ml, Streptomycin 100 U/ml, and 50 µmol/l 2ME. Medium was supplemented with Flf3-Ligand (FL) 10 ng/ml and Thrombopoietin (Tpo) 10 ng/ml as this combination of cytokines was reported to support maximal expansion of primitive umbilical cord blood cells on AFT024.
Cells were re-sorted after different cultivation time according to Forward-Scatter, Side-Scatter. Cells positive for PI were excluded and residual anti-CD34-APC was used to control efficient separation of CD34+/CD38- cells and AFT024 cells.
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