Itic cell antigen 2. doi:10.1371/journal.pone.0052902.tDengue Virus Infection in Bone MarrowFigure 4. CD61+ cells were the early cells infected by dengue virus bone marrow. Freshly aspirated bone marrows at various time points from DV infected rhesus monkeys were stained with dengue viral specific monoclonal antibody (clone 3H5) and cell lineage markers CD41, CD61, and CD14, and subjected to FACS analysis. Results revealed that viral antigen was observed early in CD61+ cells with a decreasing trend while the opposite trend was evident in CD14+ monocytic cells. doi:10.1371/journal.pone.0052902.gphagocytic dendritic cells) GSK962040 antibodies (Genway Biotec, San Diego, CA) for 1 hr. Following washing with PBS, the samples were incubated with 1:250 dilution of rabbit anti-FITC antibody conjugated to alkaline phosphatase (Sigma Aldrich, St. Louis, MO) and the signal was developed by using Vector blue alkaline phosphatase substrate kit III in the GSK2256098 cost presence of levamisole solution (Vector), an inhibitor of endogenous alkaline phosphatases. The resulting images were captured using a Zeiss microscope equipped with an Axis 5 digital camera. The number of CD41a+DV+, CD41a-DV+, BDCA+DV+, BDCA-DV+ cells were counted by assessing the number of cell surface positive or negative cells among all DV+ cells in 3? slides, or number of slides needed to assess 200 4G2 positive cells. Numbers are expressed as a percentage of total DV2+ cells. For immunofluorescent staining, smears of BM cells on glass slides were fixed with methanol for 5 minutes and rinsed with PBS. The slides were then incubated with 10 human AB serum in PBS at room temperature (RT) for 15 min followed by either mouse anti-NS1 monoclonal antibody (ab41616, Abcam, Cambridge, MA) or isotype-matched control (IgG1) antibody for 1 hr. The slides were washed and then incubated with PE-conjugated goat anti-mouse IgG antibody (eBioscience, San Diego, CA) at a dilution of 1:1000 for 1 hr. The slides were washed in PBS and then incubated with 10 normal mouse serum in PBS for 30 min at RT to block the remaining binding sites followed by the addition of FITC-conjugated mouse anti-CD61 antibody (eBioscience) for 1 hr. The slides were washed three times with PBS and mounted with DAPI mount reagent (Invitrogen, Carlsbad, CA), and images were captured using a Zeiss microscope equipped with an Axis 5 digital camera.Electron MicroscopyBM cells collected at different time intervals following infection were fixed with 2.5 glutaraldehyde in 0.1 M phosphate buffer overnight and then processed for electron microscopy by the Robert P. Apkarian Integrated Electron microscopy Core Facility Service at Emory University.histochemical staining experiments. However polyclonal anti NS-1 was used in one cell staining with human BM. The stained samples were incubated with 3-amino-9-ethylcarbazole (AEC) or diaminobenzidine (DAB) as an enzyme substrate for peroxidase followed by mounting with DAPI (Invitrogen) or counterstaining with hematoxylin. The identification of the dengue virus cell lineage consisted of staining of the preparation for dengue viral antigen in addition to a variety of cell lineage specific cell surface markers. Thus appropriate BM smears were fixed onto slides with 4 paraformaldehyde for 20 min and permeabilized with 0.2 triton X-100 for 10 min at RT. The samples were then treated with 0.6 H2O2 for 30 min to block endogenous peroxidase followed by 30-min incubation with 10 human AB serum. After two washes w.Itic cell antigen 2. doi:10.1371/journal.pone.0052902.tDengue Virus Infection in Bone MarrowFigure 4. CD61+ cells were the early cells infected by dengue virus bone marrow. Freshly aspirated bone marrows at various time points from DV infected rhesus monkeys were stained with dengue viral specific monoclonal antibody (clone 3H5) and cell lineage markers CD41, CD61, and CD14, and subjected to FACS analysis. Results revealed that viral antigen was observed early in CD61+ cells with a decreasing trend while the opposite trend was evident in CD14+ monocytic cells. doi:10.1371/journal.pone.0052902.gphagocytic dendritic cells) antibodies (Genway Biotec, San Diego, CA) for 1 hr. Following washing with PBS, the samples were incubated with 1:250 dilution of rabbit anti-FITC antibody conjugated to alkaline phosphatase (Sigma Aldrich, St. Louis, MO) and the signal was developed by using Vector blue alkaline phosphatase substrate kit III in the presence of levamisole solution (Vector), an inhibitor of endogenous alkaline phosphatases. The resulting images were captured using a Zeiss microscope equipped with an Axis 5 digital camera. The number of CD41a+DV+, CD41a-DV+, BDCA+DV+, BDCA-DV+ cells were counted by assessing the number of cell surface positive or negative cells among all DV+ cells in 3? slides, or number of slides needed to assess 200 4G2 positive cells. Numbers are expressed as a percentage of total DV2+ cells. For immunofluorescent staining, smears of BM cells on glass slides were fixed with methanol for 5 minutes and rinsed with PBS. The slides were then incubated with 10 human AB serum in PBS at room temperature (RT) for 15 min followed by either mouse anti-NS1 monoclonal antibody (ab41616, Abcam, Cambridge, MA) or isotype-matched control (IgG1) antibody for 1 hr. The slides were washed and then incubated with PE-conjugated goat anti-mouse IgG antibody (eBioscience, San Diego, CA) at a dilution of 1:1000 for 1 hr. The slides were washed in PBS and then incubated with 10 normal mouse serum in PBS for 30 min at RT to block the remaining binding sites followed by the addition of FITC-conjugated mouse anti-CD61 antibody (eBioscience) for 1 hr. The slides were washed three times with PBS and mounted with DAPI mount reagent (Invitrogen, Carlsbad, CA), and images were captured using a Zeiss microscope equipped with an Axis 5 digital camera.Electron MicroscopyBM cells collected at different time intervals following infection were fixed with 2.5 glutaraldehyde in 0.1 M phosphate buffer overnight and then processed for electron microscopy by the Robert P. Apkarian Integrated Electron microscopy Core Facility Service at Emory University.histochemical staining experiments. However polyclonal anti NS-1 was used in one cell staining with human BM. The stained samples were incubated with 3-amino-9-ethylcarbazole (AEC) or diaminobenzidine (DAB) as an enzyme substrate for peroxidase followed by mounting with DAPI (Invitrogen) or counterstaining with hematoxylin. The identification of the dengue virus cell lineage consisted of staining of the preparation for dengue viral antigen in addition to a variety of cell lineage specific cell surface markers. Thus appropriate BM smears were fixed onto slides with 4 paraformaldehyde for 20 min and permeabilized with 0.2 triton X-100 for 10 min at RT. The samples were then treated with 0.6 H2O2 for 30 min to block endogenous peroxidase followed by 30-min incubation with 10 human AB serum. After two washes w.
