Flow cytometric cell sorting of Sca-1⁺lin⁻ AM⁺ or AM⁻cells.

Low-density murine BM cells were stained with Sca-1–PE, FITC-conjugated CD3 and B220 (lin), and biotinylated antibodies recognizing either CD11a, CD43, CD44, CD49d, CD49e, or CD62L, followed by the addition of streptavidin-APC. A primary light scatter gate was constructed and used to visualize PE and FITC fluorescence (panel A). A second gate containing PE⁺ and FITC⁻ cells (Sca-1⁺lin⁻ cells, panel A) was drawn, and the APC fluorescence of these gated Sca-1⁺lin⁻ cells were visualized in a single-parameter histogram (filled histograms, panels B and C). Background APC fluorescence of Sca-1⁺lin⁻cells (open histogram, panels B and C) was determined by staining a sample with Sca-1–PE, CD3-FITC, B220-FITC, and nonspecific biotinylated antibodies followed by streptavidin-APC. Sort regions, depicted by the vertical lines and arrows in panels B and C, were created to isolate the upper and lower 30% to 40% of Sca-1⁺lin⁻ cells expressing the particular AM. For CD11a, CD43, CD49e, and CD62L, a distinct “positive” and “negative” AM fraction could be isolated (panel B), but in the cases of CD44 and CD49d, where nearly 100% of Sca-1⁺lin⁻ cells expressed these markers, the brightest and dimmest 30% to 40% of Sca-1⁺lin⁻ cells were sorted and referred to as “bright” and “dim,” respectively. To test for in vivo blocking activity of AM antibodies, a group of Sca-1⁺lin⁻ cells were sorted from each AM group while the APC signal from the AM antibody was ignored. Sort regions are shown for CD49e and CD49d as representative examples. To isolate Sca-1⁺lin⁻ cells on the basis of their expression of both CD49d and CD49e, Sca-1⁺lin⁻ cells were sorted to purity, panel A. Since these cells were FITC⁻, they were stained with FITC-labeled CD49d and biotinylated CD49e, followed by streptavidin-APC (panel D). Based on nonspecific background fluorescence, denoted by dotted box in lower left of panel D, 4 sort regions were created as follows: (1) APC⁻ FITC⁺ cells were gated and sorted as CD49e⁻ CD49d^bright; (2) APC⁺ FITC⁺ cells were sorted as CD49e⁺ CD49d^bright; (3) APC⁻FITC⁻ cells were sorted as CD49e⁻CD49d^dim; and (4) APC⁺ FITC⁻ cells were sorted as CD49e⁺ CD49d^dim (panel D).