Apc conjugated anti cd71

Single-cell suspensions of E11.5 yolk sac cells were prepared as previously described (30 ). For flow cytometric (FACS) analysis, the single-cell suspensions were labeled with PE-Cy7-conjugated anti-Ter119 (eBioscience, 25–5921) and APC-conjugated anti-CD71 (eBioscience, 17–0711). Cell data were acquired using an LSRII flow cytometer (Becton Dickson). The data analysis was performed using FlowJo.

Freshly isolated cells were washed twice in PBS (Gibco) and immunostained for 15 min with the appropriate antibody; phycoerythrin (PE)-conjugated anti-TER119 (erythroid marker), PE-conjugated Anti-cKIT (SCF receptor), PE-conjugated anti-CD41 (glycoprotein IIb), PE-conjugated anti-CD61 (glycoprotein IIIa), PE-conjugated anti-Gr-1 (granulocytic marker), PE-conjugated anti-MAC-1 (monocytic marker), APC-conjugated anti-CD71 (transferrin receptor), APC-conjugated anti-SCA-1 (primitive hematopoietic cell marker) (1:200) (eBioscience, San Diego, CA, USA). Following antibody incubation, cells were washed once in PBS and resuspended in 500 μl PBS. Cell sorting and analysis of stained cells were performed using the Becton-Dickinson FACSCalibur (BD Biosciences, San Jose, CA, USA), and the FlowJo flow cytometry analysis software (FlowJo TreeStar Inc., Ashland, OR, USA). Relative mean fluorescence intensity (MFI) values were based on the unstained population controls and calculated using the Geometric Mean statistic (average of log fluorescence). Statistical analyses were performed using the two-tailed Student's t-test, where P<0.05 was considered to indicate a statistically significant difference.