Cy5.5 rat igg 2a

For the analysis of CD49f and Sca-1 expression in the Akt transformed stem cell line, Cy5.5-CD49f (Biolegend, CA) and
pacific blue-Sca-1 antibodies (Biolegend, CA) were used. Cy5.5-rat IgG-2a (Biolegend, CA) and pacific blue-rat IgG-2a
(Biolegend, CA) were used for control antibodies. Cells (50,000) were suspended in staining buffer (5% FBS in PBS) and human
TruStain FcX™ (Biolegend, CA) was added to block Fc receptors. Cells were incubated at room temperature for 10 min
followed by incubation with antibodies or control IgG for 30 min on ice in the dark. Cells were analyzed in the RSLII flow
cytometer (BD Sciences, NJ). Proximal prostatic ductal cells were sorted by FACS (Moflo XDP, Beckman Coulter, NJ) into various
fractions (Sca-1^high, Sca-1^med/low and Sca-1^neg) according to the mean fluorescence intensity
(MFI) of Sca-1 expression by the cells after addition of PE-Sca-1 antibody (BD Sciences, NJ). PE-rat IgG-2a (BD Sciences, NJ)
was used as a negative control. Sca-1+ cells with fluorescent intensities in the upper one-third were defined as
Sca-1^high cells, the remaining Sca-1+ cells served as the Sca-1^med/low population while the
unlabeled cells served as the Sca-1^neg population. Cells (5×10⁴) were implanted with UGM
(2×10⁵) sub-renal capsule and grafts examined after 8 weeks.