Phenotypic analysis of in vitro-generated and positively selected granulocytes from B6 BM cells. (A) B6 granulocytes were generated from B6 BM cells by cultivation with G-CSF for 9 days. Enriched granulocytes were further enriched by positive selection via FACS using αmGr-1 mAb. All 3 populations were stained with mAb specific for Gr-1, NK1.1, B220, Thy1.2, and Mac-1 and analyzed with the FACSCalibur. SSC indicates side scatter. (B) Freshly isolated B6 BM cells and in vitro-propagated and FACS-sorted (αmGr-1 mAb) granulocytes were centrifuged on microscope slides and stained with May-Grünwald/Giemsa as described in “Materials and methods.” Arrows indicate polymorphonuclear cells; arrowheads, mononuclear cells. (C) Not activated B6 spleen cells and B6 spleen cells cultivated in vitro in the presence of either ConA (5 μg/mL) or ConA/ConA SN (5 μg/mL; 10% final) were subjected to FACS analysis (FACSCalibur), using mAb to Gr-1, NK1.1, B220, and/or Thy1.2. FSC indicates forward scatter. Numbers in graphs indicate the percentages of gated cells.