Apc conjugated anti ter119

Freshly isolated fetal liver cells were washed and resuspended in pre-warmed PBS with 10 μM 5-(and 6-)-chloromethyl-2’, 7’-dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Invitrogen), together with allophycocyanin (APC)–conjugated anti-Ter119 (1:200) (eBioscience) and phycoerythrin (PE)–conjugated anti-CD71 (1:200) (eBioscience) antibodies. After incubation for 30 min at 37°C, 5% CO2, the cells were washed and resuspended in PBS. Propidium iodide was added to exclude dead cells from analysis. The stained cells were immediately analyzed by flow cytometric assay.

Flow cytometric analysis of differentiation status of cultured mouse fetal erythroblasts was described previously (7 (link)). Briefly, cells were immunostained with allophycocyanin (APC)–conjugated anti-Ter119 (1:200) (eBioscience) and fluorescein isothiocyanate (FITC)–conjugated anti-CD71 (1:200) (eBioscience) antibodies for 20 min at room temperature. Propidium iodide was added to exclude dead cells from analysis. For enucleation analysis, cells were stained with 10 μg/ml Hoechst 33342 (DNA staining) in addition to stains mentioned above. Apoptosis was analyzed using an Annexin V Apoptosis Detection Kit (eBioscience) according to the manufacturer's instructions. All stained cells were analyzed on a FACSCalibur (Becton Dickinson) flow cytometer.