Expression of CD164 epitopes on Lin⁻CD34^lo/−CD38^lo/− cord blood cells.

Mononuclear cells from pools of up to 3 cord blood samples were collected, subjected to erythrocyte lysis, and Fc receptors blocked as described in “Materials and methods.” Cells were labeled with a lineage-negative cocktail of the following FITC-conjugated Mabs: CD2, CD3, CD4, CD8, CD14, CD16, CD19, CD41, CD56, CD66abce, anti-glycophorin A, with CD38-FITC, with CD34-PerCP or mIgG1-PerCP, and with the 103B2/9E10, 105A5, or isotype-matched irrelevant-control Mabs, mIgG3 or mIgM, prior to counterstaining with PE-conjugated anti-isotype–specific secondary antibodies. Cells were gated on low side scatter, low to high forward scatter, and lineage-negative CD38^lo/−(Lin⁻CD38^lo/−) fluorescein gates (upper left dot plot) according to Bhatia et al.29 The CD34-PerCP and CD164 epitope/PE-conjugated anti-mIgG3 or mIgM fluorescence was determined against mIgG1-PerCP and mIgG3 or mIgM/PE-conjugated anti-mIgG3 or mIgM-matched controls. Cells were analyzed on a FACSCalibur and 400 × 103to 500 × 10³ events stored as listmode data files per sample. The upper right dot plots show the forward and side scatter distribution of Lin⁻CD38^lo/− cells after CD34⁺ or CD34⁻ cell gating. The lower dot plots demonstrate the staining of Lin⁻CD38^lo/− cells labeled with isotype-matched control Mabs or with CD164 and CD34 Mabs. The median fluorescence intensity values for mIgG3- and mIgM-labeled Lin⁻CD34^lo/−CD38^lo/−cells were 3 ± 1 and 2 ± 1, respectively, and Lin⁻CD34⁺CD38^lo/− cells were 1 ± 2 and 2 ± 1, respectively.