![Figure 4. LSECtin expression on the cell surface of monocyte-derived dendritic cells and thymic-cell subsets. (A) Cell extracts were obtained from monocytes along the MDDC differentiation pathway by treating them with GM-CSF plus IL-4 for the indicated periods of time. In parallel, extracts were obtained from GM-CSF-generated macrophages after a 48-hour treatment with medium (MDM), IL-4 (AAMØ), or interferon-γ (CAMØ). In all cases, 10 μg of each lysate was subjected to Western blot for detection of LSECtin with the ADS-1 polyclonal antiserum and β-actin expression determined in parallel for loading control purpose. (B) Cell surface expression levels of LSECtin in monocyte-derived dendritic cells from three independent donors as determined with the antiLSECtin SOTO1 monoclonal antibody. The supernatant from the P3 × 63 myeloma was used as negative control (X63). (C) MDDC were allowed to differentiate on glass coverslips, fixed (paraformaldehyde 2%), washed, and incubated with either a polyclonal antiserum against LSECtin (ADS-1) or preimmune serum. After washing, cells were incubated with a 1:500 dilution of Alexa 488-labeled goat anti-rabbit IgG (F(ab′)2) antiserum. Nuclei were counterstained with 4′,6-diamidino-2-fenilindol diclorhidrato (DAPI). Coverslips were mounted in Dako Cytomation fluorescent mounting medium (Dako, Glostrup, Denmark) and representative fields photographed through an HCX PL APO lens (63.0 × 1.40 oil objective), with a 1.400000 numerical aperture on a TCS-SP2-ADBS confocal laser LEICA scanning system (LEICA MICROSYSTEMS GmbH, Wetzlar, Germany). (D) Expression of LSECtin in human thymic cell populations. Human thymic cell preparations were depleted of thymocytes by centrifugation on Ficoll after rosetting with sheep erythrocytes. The resulting population was then stained with either PE-labeled anti-CD123 and FITC-labeled BDCA2 or with FITC-labeled anti-CD14 and PE-labeled anti-CD13 antibodies. The BDCA2+ CD123+ (plasmacytoid DC [pDC]), CD14+ CD13+ (macrophages [MØ]), and CD14−CD13+ (myeloid DC [MyDC]) subsets (left panels) were simultaneously stained with the anti-LSECtin SOTO1 monoclonal antibody followed by incubation with an APC-labeled goat anti-mouse antiserum (right panels).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/109/12/10.1182_blood-2006-09-048058/4/zh80120701780004.jpeg?Expires=1730480905&Signature=C7-oSbK~rad1dMHAvEgv0wkN3~IZv1vsOE2e7m1yaLcyYb9Y7b0r2EdRhD79XVVY22OONcMTRJh~8Pmf0cHA5hZFd1eAeBPQRUPu6j~Jx2X7buz9pbW0BoeWLOu5dzub02CJSijLwiXYw0DG6weR6AsiM-IJ72kkDV5gVxTrqndk4-v~iiCj85X6fxrfn6ATO-QXKVgZDyQPSy0YYa39ASAiR8tjneKvvaKAJHMw3Lt7Zv4uKSCIMOdzqVq2hVXOK9QKEEC~uUOAWgKPvQuBvni-sUJTFqKxwthZvKGn8dquW54IqxHZxVS4qPopYKQ4L11dg~U~KvIHN4dVxvn4KA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
LSECtin expression on the cell surface of monocyte-derived dendritic cells and thymic-cell subsets. (A) Cell extracts were obtained from monocytes along the MDDC differentiation pathway by treating them with GM-CSF plus IL-4 for the indicated periods of time. In parallel, extracts were obtained from GM-CSF-generated macrophages after a 48-hour treatment with medium (MDM), IL-4 (AAMØ), or interferon-γ (CAMØ). In all cases, 10 μg of each lysate was subjected to Western blot for detection of LSECtin with the ADS-1 polyclonal antiserum and β-actin expression determined in parallel for loading control purpose. (B) Cell surface expression levels of LSECtin in monocyte-derived dendritic cells from three independent donors as determined with the antiLSECtin SOTO1 monoclonal antibody. The supernatant from the P3 × 63 myeloma was used as negative control (X63). (C) MDDC were allowed to differentiate on glass coverslips, fixed (paraformaldehyde 2%), washed, and incubated with either a polyclonal antiserum against LSECtin (ADS-1) or preimmune serum. After washing, cells were incubated with a 1:500 dilution of Alexa 488-labeled goat anti-rabbit IgG (F(ab′)2) antiserum. Nuclei were counterstained with 4′,6-diamidino-2-fenilindol diclorhidrato (DAPI). Coverslips were mounted in Dako Cytomation fluorescent mounting medium (Dako, Glostrup, Denmark) and representative fields photographed through an HCX PL APO lens (63.0 × 1.40 oil objective), with a 1.400000 numerical aperture on a TCS-SP2-ADBS confocal laser LEICA scanning system (LEICA MICROSYSTEMS GmbH, Wetzlar, Germany). (D) Expression of LSECtin in human thymic cell populations. Human thymic cell preparations were depleted of thymocytes by centrifugation on Ficoll after rosetting with sheep erythrocytes. The resulting population was then stained with either PE-labeled anti-CD123 and FITC-labeled BDCA2 or with FITC-labeled anti-CD14 and PE-labeled anti-CD13 antibodies. The BDCA2+ CD123+ (plasmacytoid DC [pDC]), CD14+ CD13+ (macrophages [MØ]), and CD14−CD13+ (myeloid DC [MyDC]) subsets (left panels) were simultaneously stained with the anti-LSECtin SOTO1 monoclonal antibody followed by incubation with an APC-labeled goat anti-mouse antiserum (right panels).
