Figure 1.

Isolation of CXCR4⁺ and CXCR4⁻ subfractions within primitive human hematopoietic populations. (A) A representative FACS analysis of human neuronal (i), muscle (ii), and CB cells (Lin⁻) (iii), stained with either 12 g5 or 44717.111 anti-human CXCR4 antibodies conjugated to PE. The percentage of CXCR4⁺ cells detected is given above each positive gate as the mean ± SEM of four (neural), two (muscle), and six (hematopoietic) different samples. CB Lin⁻ cells stained with mouse IgG1 served as isotype to establish sorting gates for the purification of CXCR4⁻ and CXCR4⁺ hematopoietic subpopulations indicated as R1 and R2, respectively (iii). Subpopulations of CD34⁺ CD38⁻Lin⁻ cells were isolated by using the R3 sorting gate (iv). (B) CD34⁺ CD38⁻Lin⁻ cells stained with IgG1-PE were analyzed by FACS and confocal to serve as a control (i). Fluorescence of control (i) was compared with purified CD34⁺ CD38⁻Lin⁻CXCR4⁻ (R1 and R3) and CD34⁺ CD38⁻Lin⁻CXCR4⁺ (R2 and R3) cells, a measure of sort purity (ii and iii). Selected CXCR4⁻ cells were restained for CXCR4 by using 12 g5 or 44717.111 and reanalyzed to verify the absence of any CXCR4⁺ cells (iv). For confocal microscopy, primitive CD34⁺ CD38⁻Lin⁻ cells stained with isotype control, CD34⁺ CD38⁻Lin⁻CXCR4⁻, and CD34⁺ CD38⁻Lin⁻CXCR4⁺ cells were isolated by FACS and visualized with a Zeiss LSM-410 confocal microscope at random z-planes. Representative photographs depicting the PE and FITC fluorescence from CD34⁺ CD38⁻Lin⁻ isotype control (i), CD34⁺ CD38⁻Lin⁻CXCR4⁻ (ii), and CD34⁺ CD38⁻Lin⁻CXCR4⁺ (iii) cells are presented along side each corresponding population reanalyzed by FACS (n = 4).