described previously[52]. Briefly, marrow cells prepared from the CD45.1 mice were first depleted of red blood cells using ACK solution (0.15 M ammonium and 0.01 M potassium carbonate). Red cell-depleted bone marrow cells were then enriched for c-Kit+ cells by positive selection using purified anti-c-Kit antibody (clone ACK45; BD Pharmingen) followed by incubation with goat anti- rat magnetic beads from Miltenyi Biotec. C-Kit-enriched bone marrow cells were subsequently stained with a cocktail of primary monoclonal antibodies consisting of biotin-conjugated anti-CD3 (clone 145-2C11), anti-CD4 (clone RM4-5), anti-CD5 (clone 53– 7.3), anti-CD8a (clone 53–6.7), anti-CD11b (clone M1/70), anti- B220 (clone RA3-6B2), anti-Gr-1 (clone RB6-8C5), and anti- TER-119 (clone TER-119); FITC-conjugated anti-Thy1.1 (clone OX-7); PE-conjugated anti-Sca-1 (clone E13-161.7); and allophy- cocyanin-conjugated anti-c-Kit (clone 2B8); followed by secondary staining with streptavidin-Cy-Chrome (all purchased from BD Pharmingen). Propidium iodide (1 mg/mL; Sigma, St Louis, MO) was added to the cell suspension to exclude dead cells before cell sorting. The stained samples were sorted for c-Kit + Thy1.1low Lin2/lowSca-1+ hematopoietic stem cells (KTLS) cells using a dual laser fluorescence activated cell sorter (FACS) Vantage SE (Becton Dickinson, San Jose, CA). The recipient CD45.2 mice were given total body irradiation using a cesium irradiator. The mice received 10.5 Gy irradiation in the first 2 sets of experiments and 9.5 Gy in the third set of experiment. Within 4 hours after irradiation, the CD45.2 mice were injected via tail vein with the sorted KTLS hematopoietic stem cells (250 KTLS cells per mouse). AMD3100 administration The transplanted mice were injected subcutaneously with PBS mock control or AMD3100 at 5 mg/Kg body weight in a volume of 100 ml every Monday, Wednesday and Friday beginning at day +2 post transplant until day +56. Animal survival was monitored daily. Measurement of hematological recovery To determine peripheral blood hematological recovery and to quantify various cell subsets, blood samples were obtained using heparinized capillary tubes at the indicated time points. Fifty micro-liters of blood were stained with monoclonal antibodies for 15 minutes at room temperature. The stained whole-blood samples were then processed in BD FACSTM Lysing solution to lyse red blood cells. Flow-Count fluorospheres (50 mL; Beckman- Coulter) were added before flow cytometric analysis. Stained cells were analyzed using BD FACSCantoTM (BD Biosciences, San Jose, CA). The absolute counts were calculated using the following formula: Absolute count (cells/mL blood) = (Total number of cells counted/Total number of fluorospheres counted) x Flow-Count fluorosphere concentration. Hemoglobin concentration and plate- let counts were measured using a HemavetH counter (Drew Scientific, Inc, Dallas, Texas) as per manufacturer’s instruction. To determine marrow and spleen hematopoietic stem/progen- itor cell recovery, the transplanted recipient mice were sacrificed at day +65, and 2 femurs, 2 tibias and the spleen were harvested. Several analyses were performed including histological examina- tion, nucleated marrow cell counts, colony forming unit assay (CFUs), and stem cell quantification. One femur and one tibia were used for histological examination. The femur and tibia were fixed in 10% buffered neutral formalin (VWR International, West Chester, PA). Bone was decalcified, stained with H&E, and examined by microscopy. The other femur and tibia marrow cavities were flushed, and marrow cells were resuspended in 4 ml of RPMI1640 containing 10% fetal calf sera. The total nucleated AMD3100 Enhances Engraftment cells of the femur and the tibia were counted using the HemavetH counter. To determine the frequency of colony forming units- granulocytes and megakaryocytes (CFUs-GM), marrow cells from individual mice were plated in MethocultH GF 3434 (16105 cells/ dish, triplicate dishes per individual mouse) as per manufacturer’s instruction (StemCell Technology, Vancouver, Canada). The number of CFUs-GM was counted at day 12. To determine the numbers of donor- derived CD45.1+ c-kit+ Lin2 Sca-1+ (KLS) hematopoietic stem cells and recipient-origin CD45.2+ KLS cells, the marrow cell suspension was stained with PE-conjugated CD45.1 antibody, FITC-conjugated CD45.2, APC-labeled c-Kit antibody, PE-Cy5 conjugated Sca-1 and biotin-conjugated anti- CD3 (clone 145-2C11), anti-CD4 (clone RM4-5), anti-CD5 (clone 53-7.3), anti-CD8a (clone 53-6.7), anti-CD11b (clone M1/70), anti-B220 (clone RA3-6B2), anti-Gr-1 (clone RB6-8C5), and anti- TER-119 (clone TER-119); followed by secondary staining with streptavidin-Cy-Chrome. The absolute KLS stem cell number in one femur and one tibia was calculated multiplying total number of nucleated marrow cells with % KLS cells on flow cytometry analysis. The absolute cell numbers of recipient-origin stem cells (CD45.2+ KLS cells) and donor derived stem cells (CD45.1+ KLS cells) in the spleen were similarly determined. Cytokines/chemokines measurement Lethally irradiated CD45.2 mice were transplanted with KTLS cells and treated with PBS or AMD3100 as described above. At day +7 post transplantation, the mice were bled and plasma prepared. The cytokines/chemokines (pg/ml) was measured using Bioplex-mouse 23 Plex Cytokines Kit as per manufacturer’s instruction (Bio-Rad Laboratories, Hercules, CA). These cyto- kines/chemokines are IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-17, Eotaxin, G- CSF, GM-CSF, IFN-l, keratinocyte chemoattractant (KC), monocyte chemoattractant protein-1 (MCP-1), MIP-1a, MIP-1b, TNF-a, and RANTES. Measurement of colony forming unit of spleen at day +9 Lethally irradiated CD45.2 mice (10.5 Gy) were injected with whole marrow cells from CD45.1 mice (16105 marrow cells per mouse). The recipient mice were injected subcutaneously with PBS buffer or AMD3100 at 5 mg/kg every other day beginning at day +2. At day +9, the mice were sacrificed and the spleen was harvested and fixed in Bouin’s solution. The colony forming units of spleen (CFUs-spleen) were counted. In vivo stem cell mobilization and homing studies shortly after transplantation Lethally irradiated C57BL/6 CD45.2 Thy1.2 mice (10.5 Gy) were injected via tail vein with sorted KTLS hematopoietic stem cells (2500 cells/mouse) obtained from C57BL/Ka Thy1.1 donor mice. At 48 hours after transplantation, recipient mice were injected subq with a single dose of AMD3100 at 5 mg/kg or PBS control. Twenty-four hours after this injection, the mice were sacrificed, and the 2 femurs and the spleen were harvested. The total nucleated cells in the 2 femurs and in the spleen were measured using the HemavetH counter. The absolute cell numbers of recipient-origin stem cells (CD45.2+ KLS cells) and donor derived stem cells (CD45.1+ KLS cells) in the spleen and in the marrow were determined, as described above. Marrow CFUs-GM and Bursting forming units of erythrocytes (BFUs-E) were measured using MethocultH GF 3434 (16105 cells/dish, triplicate dishes per individual mouse) as per manufacturer’s instruction (StemCell Technology, Vancouver, Canada). Percent donor stem PLoS ONE | www.plosone.org 12 June 2010 | Volume 5 | Issue 6 | e11316