Using a microcavity array system [22,23,24] as described previously [22]. Briefly, blood samples

Using a microcavity array system [22,23,24] as described previously [22]. Briefly, blood samples (,20 mL) from the tail vein of transplanted NOG mice were stained with Hoechst 33342 (Life Technologies, Carlsbad, CA) and fluorophore-labeled antibodies. For analysis of Hu-NOG mice, FITC-conjugated antihCD45 monoclonal antibodies (mAbs) and PE-conjugated antimCD45 mAbs (both from BD Biosciences, San Jose, CA) were used. For analysis of Mo-NOG mice, FITC-conjugated antimCD45.2 mAbs and PE-conjugated anti-mCD45.1 mAbs (both from BD Biosciences) were used. Stained blood samples were passed through the microcavities with negative pressure, and only leucocytes were captured. Then, a whole image of the cell array area was obtained using an IN Cell Analyzer 2000 (GE Healthcare Life Sciences, Little Chalfont, UK). The number and rate of host and donor-derived leukocytes was determined from the scanned fluorescence signal of arrayed leukocytes. On the basis of body weight, the sum 25331948 of DBeQ leukocyte counts, and the rates of leukocyte chimerism, Hu-NOG mice were divided into 5 groups of 9?0 mice per group without significant differences between each group. The rates of leukocyte chimerism were calculated as the percentage of donor-derived leukocytes in the total leukocyte population (the sum of donor- and host-derived leukocytes). Mo-NOG mice were divided into 4 groups of 8 mice each.Materials and Methods Cells and MiceTransplanted human CD34+ cells isolated from 18325633 cord blood were purchased from Lonza (Lot: OF4563, Basel, Switzerland) and cryopreserved in liquid nitrogen prior to use. Transplanted mouse Lin2 bone marrow cells were prepared from the femurs of 6-week-old C57BL/6J mice. Bone marrow cells were collected by excising and crushing the epiphysis and metaphysis with a mortar and by pushing a needle through the diaphysis. Lin2 cells were further purified using a Lineage Cell Depletion Kit (Miltenyi Biotec, Bisley, UK). Fresh Lin2 bone marrow cells were used in experiments. As hosts for cell transplantation, immunodeficient NOD/Shi-scid/IL-2Rcnull (NOG) mice (6-week-old, male) were obtained from the Central Institute for Experimental order JRF 12 Animals (Kawasaki, Japan). Mice were housed in a specific pathogen-free facility in autoclaved polycarbonate cages and fed sterile food and water ad libitum. In addition, NOG mice were maintained on neomycin-polymyxin B in their drinking water.Administration of BenzenePublished epidemiological research regarding short-term exposure to benzene has shown that the lowest-observed adverse effect level (LOAEL) of benzene-induced hematotoxicity based on decreasing leukocyte counts in the peripheral blood, is 60 ppm [25]. When inhalation exposure levels are converted into oral administration levels, 60 ppm is equivalent to 30 mg benzene/kgb.w./day (conversion conditions are as follows: respiratory volume, 20 m3/day; absorptivity, 50 ; body weight, 70 kg) [26]. Benzene toxicity depends on the amount absorbed and not the site of absorption [26,27]. Therefore, in the present study, 0, 10, 30, 100, and 300 mg/kg-b.w. benzene, suspended in corn oil, were administered by gavage to Hu-NOG mice daily for 2 weeks, starting at about 4 months after transplantation. In the case of MoNOG mice, the amounts of benzene administered were 0, 30, 100, and 300 mg/kg-b.w./day. Because mouse cells have lowerFigure 1. Schematic of the method. After a 2-week quarantine and acclimatization period, human CD34+ cells or mouse Lin2 bone marrow cells were i.Using a microcavity array system [22,23,24] as described previously [22]. Briefly, blood samples (,20 mL) from the tail vein of transplanted NOG mice were stained with Hoechst 33342 (Life Technologies, Carlsbad, CA) and fluorophore-labeled antibodies. For analysis of Hu-NOG mice, FITC-conjugated antihCD45 monoclonal antibodies (mAbs) and PE-conjugated antimCD45 mAbs (both from BD Biosciences, San Jose, CA) were used. For analysis of Mo-NOG mice, FITC-conjugated antimCD45.2 mAbs and PE-conjugated anti-mCD45.1 mAbs (both from BD Biosciences) were used. Stained blood samples were passed through the microcavities with negative pressure, and only leucocytes were captured. Then, a whole image of the cell array area was obtained using an IN Cell Analyzer 2000 (GE Healthcare Life Sciences, Little Chalfont, UK). The number and rate of host and donor-derived leukocytes was determined from the scanned fluorescence signal of arrayed leukocytes. On the basis of body weight, the sum 25331948 of leukocyte counts, and the rates of leukocyte chimerism, Hu-NOG mice were divided into 5 groups of 9?0 mice per group without significant differences between each group. The rates of leukocyte chimerism were calculated as the percentage of donor-derived leukocytes in the total leukocyte population (the sum of donor- and host-derived leukocytes). Mo-NOG mice were divided into 4 groups of 8 mice each.Materials and Methods Cells and MiceTransplanted human CD34+ cells isolated from 18325633 cord blood were purchased from Lonza (Lot: OF4563, Basel, Switzerland) and cryopreserved in liquid nitrogen prior to use. Transplanted mouse Lin2 bone marrow cells were prepared from the femurs of 6-week-old C57BL/6J mice. Bone marrow cells were collected by excising and crushing the epiphysis and metaphysis with a mortar and by pushing a needle through the diaphysis. Lin2 cells were further purified using a Lineage Cell Depletion Kit (Miltenyi Biotec, Bisley, UK). Fresh Lin2 bone marrow cells were used in experiments. As hosts for cell transplantation, immunodeficient NOD/Shi-scid/IL-2Rcnull (NOG) mice (6-week-old, male) were obtained from the Central Institute for Experimental Animals (Kawasaki, Japan). Mice were housed in a specific pathogen-free facility in autoclaved polycarbonate cages and fed sterile food and water ad libitum. In addition, NOG mice were maintained on neomycin-polymyxin B in their drinking water.Administration of BenzenePublished epidemiological research regarding short-term exposure to benzene has shown that the lowest-observed adverse effect level (LOAEL) of benzene-induced hematotoxicity based on decreasing leukocyte counts in the peripheral blood, is 60 ppm [25]. When inhalation exposure levels are converted into oral administration levels, 60 ppm is equivalent to 30 mg benzene/kgb.w./day (conversion conditions are as follows: respiratory volume, 20 m3/day; absorptivity, 50 ; body weight, 70 kg) [26]. Benzene toxicity depends on the amount absorbed and not the site of absorption [26,27]. Therefore, in the present study, 0, 10, 30, 100, and 300 mg/kg-b.w. benzene, suspended in corn oil, were administered by gavage to Hu-NOG mice daily for 2 weeks, starting at about 4 months after transplantation. In the case of MoNOG mice, the amounts of benzene administered were 0, 30, 100, and 300 mg/kg-b.w./day. Because mouse cells have lowerFigure 1. Schematic of the method. After a 2-week quarantine and acclimatization period, human CD34+ cells or mouse Lin2 bone marrow cells were i.