1. The Chemokine Receptor CXCR4 Is Required for the Retention of B Lineage and Granulocytic Precursors within the Bone Marrow Microenvironment 
Qing Ma, Dan Jones, Timothy A Springer 
Immunity 
Volume 10, Issue 4, Pages (April 1999)
DOI: /S (00)

2. Figure 1
Immunofluorescent Flow Cytometry of B Lymphoid and Myeloid Cells in E18.5 Fetal Blood and Liver
The percentages of total cells within the box are shown. The data are representative of six independent experiments. Genotypes are +/+, wild-type control, and −/−, CXCR4-deficient embryos. (A) B lineage cells. Pro-B cells (B220+CD43+) within the lymphocyte gate are boxed. (B) Myeloid lineage cells. Myeloid lineage cells were identified by Gr-1 and CD11b mAbs.
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3. Figure 2
Cell Populations in Peripheral Blood of Mice 4 Weeks Post Transplant
Blood samples were collected from four different mice. Total white blood cells (WBC) were determined by Coulter counter. Donor-derived B cells (CD45.2+B220+) and granulocytes (CD45.2+Gr-1+) were determined by flow cytometry. Genotypes refer to the source of the donor fetal livers.
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4. Figure 3 B Lymphopoiesis in Reconstituted Mice
In all experiments, cells were stained either with CD45.2 and B220 mAbs for donor-derived B cells (CD45.2+B220+) or with B220, CD43, and IgM mAbs for pro-B (B220+CD43+IgM−), pre-B (B220+CD43−IgM−), and IgM+ B (B220+CD43−IgM+) cell subpopulations; concordant results were obtained. The lower two panels in (A) and (D) show cells triple-stained with mAb to B220, CD43, and IgM. In all experiments with bone marrow cells from animals 4 weeks after transplant and after culture of these cells in vitro, B220+ cells were more than 90% CD Genotypes above each column refer to the source of donor fetal livers. The data are representative of four independent experiments. The averages and SD values reported in the text are for all experiments. (A) Immunofluorescent flow cytometry of bone marrow. The percentages of total nucleated cells are shown in the upper panel. The percentages of cells within lymphoid side and forward angle scatter gates are shown in the middle and lower panels. (B and C) In vitro bone marrow culture under Whitlock-Witte conditions for 5 days in the presence (B) or absence (C) of IL-7. (D) B cell subpopulations in peripheral blood. The boxed areas in the two lower panels in (D) contain cells with pro-B and pre-B cell phenotypes, which are B220+IgM− and are differentiated as CD43+ or CD43−, respectively. (E and F) Immunofluorescent flow cytometry of bone marrow cells (E) and peripheral blood leukocytes (F) in mice 4 months post transplant.
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5. Figure 4
In Vitro Culture of IgM− B Cells Isolated from the Peripheral Blood of Mice 4 Weeks Post Transplant with CXCR4-Deficient Fetal Liver Cells
The B220+IgM− cells in peripheral blood purified by cell sorting were analyzed by flow cytometry (A). The purified cells were cultured on ST2 stromal cells under Whitlock-Witte conditions (Whitlock and Witte 1982) with (B) and without (C) IL-7. Cells were collected 10 days after culture and analyzed by flow cytometry. The data represent two independent culture experiments. In cultures without IL-7, stromal cells were washed off the well and contributed to the B220− population. The B220+ cells were more than 98% CD45.2+.
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6. Figure 5 Myelopoiesis in Mice 4 Weeks Post Transplant
Genotypes refer to the source of donor fetal livers. (A) Analysis of myeloid cells in bone marrow by flow cytometry. The yields of cells per femur from wild-type and CXCR4-deficient transplants were similar and ranged from 2.4 × 107 to 4.0 × 107 from a total of eight femurs from four sets of independent experiments. The percentages of Gr-1+CD45.2+ population (boxed) of total cells are shown. (B) Bone marrow cytospin samples stained with Wright-Giemsa. Arrows indicate myeloid elements (magnification 1000×). (C) Bone marrow sections stained with hematoxylin/eosin (magnification 400×). Arrows indicate myeloid elements. (D) Immunofluorescent flow cytometry of peripheral granulocytes. The upper panel dot plots display the cells within a forward and side scatter gate that was set broadly to include granulocytic cells. The Gr-1+CD45.2+ cells are boxed, and the percentages of these populations of the total nucleated cells are shown. The lower panel dot plots display the forward scatter and side scatter of the cells within the box in the corresponding upper panel. For reference, the circle including the scatter distribution of the wild-type granulocytes is shown in both plots. (E) Blood cytospin samples stained with Wright-Giemsa (W-G) and for chloroacetate esterase activity (CAE) (magnification 1000×). In the W-G stained smears, arrowheads indicate segmented granulocytes and thin arrows indicate metamyelocytes/ring forms. In the CAE stained smears, thick arrow indicates myelocyte.
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7. Figure 6
Immunofluorescent Flow Cytometry of B and T Lymphocytes in Lymph Node and Spleen from Mice 4 Months Post Transplant with CXCR4-Deficient Fetal Liver Cells
The dot plots display cells within the lymphocyte gate. The percentages of total gated cells within the box are shown.
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8. Figure 7
Lymphocyte Localization in Lymph Node and Spleen from Mice 4 Months Post Transplant with CXCR4-Deficient Fetal Liver Cells
(A–C) Sections of lymph node stained with hematoxylin/eosin (A), B220 mAb (B), and CD3 mAb (C). (D and E) Sections of spleen stained with hematoxylin/eosin (D) and B220 mAb (E). (F) Section of Peyer’s patch stained with hematoxylin/eosin. M, mantle zone of follicle; GC, germinal center; Foll, follicle; PALS, periarteriolar lymphoid sheath; and RP, red pulp.
Immunity  , DOI: ( /S (00) )