GRP94 Regulates Stem Cell-Niche Interactions Figure 11. Grp94 KO mice displayed altered myeloid and lymphoid differentiation. A). Complete blood count of peripheral blood from WT (n = 31) and cKO (n = 37) mice. B) Representative Wright-Giemsa staining of blood smear with tail peripheral blood from WT and cKO mice. Scale bar represents 500 mm. C) Total thymus cell number (left) and total left and right axillary lymph nodes cell number (right) from WT and cKO mice (n = 7 for each group). D) Representative flow cytometric analysis of splenocytes from WT and cKO mice using lineage markers Gr-1 and CD3 (left), F4/80 and B220 (right). E) Quantitation of (D) from WT (n = 4) and cKO (n = 7) mice. F) Representative flow cytometric analysis with BM cells using lineage markers Gr-1 and B220 (left) and CD4 and CD8a (right). G) Quantitation of (F). Gr-1 and B220 (n = 7 for WT and n = 9 for cKO mice); CD4 and CD8a (n = 7 for each genotype). All data are presented as mean 6 s.e., ***p,0.001. doi:10.1371/journal.pone.0020364.g011 Splenocytes were collected following disaggregation of the spleens in DPBS, with red blood cells (RBCs) lysed in lysing buffer (BD Biosciences) for 15 min and then filtered through nylon screen to obtain single cell suspension. Peripheral blood samples were collected by tail bleeding, with RBCs lysed in lysing buffer twice for 15 min. Cells from mouse tissue [BM, spleen and peripheral blood mononuclear cells (MNCs)] were resuspended in DPBS with 0.5% BSA and 0.1% sodium azide for antibody staining. To study hematopoietic stem and progenitor cells, Lineage (Lin) which consists of B220 (RA3-6B2), TER119 (TER119), CD4 (RM4-5), CD8a (53-6.7), Gr-1 (RB6-8C5), and Mac-1 (WT.5), c-Kit (2B8), Sca-1 (D7), Flk2 (A2F10.1) CD34 (RAM34), IL7Ra (SB/199) and FccRII/III (2.4G2, all antibodies from BD Pharmingen) were used. To study different lineages of blood cells, Gr-1, F4/80 (Invitrogen), B220, CD4, CD8a and CD3 (1F4) were used. CD45.1 (A20) and CD45.2 (104, both from BD Pharmingen) were used for BM transplantation analysis. Integrin a4 and a5 were detected using CD49d (R1-2, eBioscience) and CD49e (5H10-27, BD Pharmingen). 36106-16107 BM or spleen cells, or MNCs from 200 mL peripheral blood were incubated with antibodies for 30 min, washed twice with DPBS with 0.5% BSA and 0.1% sodium azide, and then resuspended in DPBS. Dead cells were excluded by 7AAD staining (BD Pharmingen). Cell population analysis was performed on BD FACS LSR II. Fluorescence activated cell sorting Mouse whole BM cells resuspended in DPBS were enriched for c-Kit+ cells using CD117 MicroBeads (Miltenyi Biotec, Auburn, CA) according to manufacturer’s instructions. c-Kit+ cells were stained with anti-mouse c-Kit (Miltenyi Biotec), anti-mouse Sca-1, and Lin antibody cocktail as described above. Primitive hemato- poietic stem and progenitor cells were purified using a FACSAria flow cytometer (Becton Dickinson) based on established cell surface phenotypes. LSK cells were sorted for the competitive repopulation and adhesion assays; LK cells were purified for the in vivo homing and lodgment assay. Cell cycle analysis 16107 whole BM cells were incubated with 10 mg/ml Hoechst 33342 (Sigma-Aldrich) at 37uC for 45 min, then stained with primitive hematopoietic cell antibodies (Lin, c-Kit and Sca-1) as described above. The stained cells were resuspended in 10% neutral buffered formalin (BDH Chemicals) and incubated at 4uC overnight. To stain for RNA content, Pyronin Y (Polysciences PLoS ONE | www.plosone.org 12 May 2011 | Volume 6 | Issue 5 | e20364