Figure 5
Figure 5. C1q colocalizes with CD40 after CD40 ligation on DCs. (A) GM-CSF DCs were incubated with exogenous C1q (50 μg/mL) and binding of C1q was detected by staining with FITC-conjugated anti-C1q antibodies (gray histogram) using flow cytometry. The black histogram represents control staining with the second step reagent only. (B) CD4+ T cells isolated from spleen and lymph nodes of WT mice were incubated with C1q (50 μg/mL) and bound C1q (gray histogram) was detected as described for panel A. The black profile represents control staining with the second step reagent only. (C) GM-CSF DCs were stimulated with LPS (10 ng/mL) for 48 hours to induce their maturation. The DCs were then incubated with C1q (50 μg/mL). Bound C1q was detected by staining with FITC-conjugated anti-C1q antibodies (green) and CD40 expression was detected using anti-CD40 biotinylated antibody and streptavidin-Alexafluor 633 (red). The nuclei were counterstained with Hoechst 33342 (blue) and analysis was done by confocal microscopy. Imaging was done using a Leica SP2 upright confocal microscope (Heidelberg, Germany). (D) GM-CSF DCs were loaded with HY peptide and then cocultured with Marilyn T cells for 48 hours to induce CD40 ligation on DCs. Then the DCs were incubated with C1q (50 μg/mL) and stained as described for panel C to analyze C1q binding by confocal microscopy. Results are representative of 2 independent experiments performed with DCs from different mice. (E) GM-CSF DCs or GM-CSF/IL-4 DCs were evaluated for the expression of calreticulin (CRT) by staining with anticalreticulin antibodies followed by FITC-conjugated secondary antibodies. (F) CRT expression was evaluated using confocal microscopy on DCs stained with anticalreticulin antibodies and FITC-conjugated secondary antibodies. (G) GM-CSF/IL-4 DCs were cultured alone or in the presence of LPS for 48 hours and CRT expression was assessed. (H) GM-CSF/IL-4 DCs were cocultured with Marilyn T cells in the presence or absence of peptide for 48 to 72 hours and stained with anti-CD11c and anticalreticulin antibodies. (I) DCs pulsed with male or female apoptotic thymocytes (DC + M apo and DC + F apo, respectively) were cultured with Marilyn T cells for 48 hours in the presence or absence of blocking anticalreticulin antibodies and supernatants tested for IL-2.

C1q colocalizes with CD40 after CD40 ligation on DCs. (A) GM-CSF DCs were incubated with exogenous C1q (50 μg/mL) and binding of C1q was detected by staining with FITC-conjugated anti-C1q antibodies (gray histogram) using flow cytometry. The black histogram represents control staining with the second step reagent only. (B) CD4+ T cells isolated from spleen and lymph nodes of WT mice were incubated with C1q (50 μg/mL) and bound C1q (gray histogram) was detected as described for panel A. The black profile represents control staining with the second step reagent only. (C) GM-CSF DCs were stimulated with LPS (10 ng/mL) for 48 hours to induce their maturation. The DCs were then incubated with C1q (50 μg/mL). Bound C1q was detected by staining with FITC-conjugated anti-C1q antibodies (green) and CD40 expression was detected using anti-CD40 biotinylated antibody and streptavidin-Alexafluor 633 (red). The nuclei were counterstained with Hoechst 33342 (blue) and analysis was done by confocal microscopy. Imaging was done using a Leica SP2 upright confocal microscope (Heidelberg, Germany). (D) GM-CSF DCs were loaded with HY peptide and then cocultured with Marilyn T cells for 48 hours to induce CD40 ligation on DCs. Then the DCs were incubated with C1q (50 μg/mL) and stained as described for panel C to analyze C1q binding by confocal microscopy. Results are representative of 2 independent experiments performed with DCs from different mice. (E) GM-CSF DCs or GM-CSF/IL-4 DCs were evaluated for the expression of calreticulin (CRT) by staining with anticalreticulin antibodies followed by FITC-conjugated secondary antibodies. (F) CRT expression was evaluated using confocal microscopy on DCs stained with anticalreticulin antibodies and FITC-conjugated secondary antibodies. (G) GM-CSF/IL-4 DCs were cultured alone or in the presence of LPS for 48 hours and CRT expression was assessed. (H) GM-CSF/IL-4 DCs were cocultured with Marilyn T cells in the presence or absence of peptide for 48 to 72 hours and stained with anti-CD11c and anticalreticulin antibodies. (I) DCs pulsed with male or female apoptotic thymocytes (DC + M apo and DC + F apo, respectively) were cultured with Marilyn T cells for 48 hours in the presence or absence of blocking anticalreticulin antibodies and supernatants tested for IL-2.

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