Figure 6
Binding of anti-Sm to neuronal cells. (A) After fixation with 1% paraformaldehyde, SK-M-NC cells and Neuro2a cells were reacted with murine monoclonal anti-Sm or control murine immunoglobulin G (IgG3) (5 μg/ml), followed by counterstaining with fluorescein isothiocyanate (FITC)-conjugated goat F(ab’)2 anti-mouse IgG and with propidium iodide (PI). The cells were then analyzed by flow cytometry. Representative FITC stainings on viable cells gated by negative staining with PI are shown. The percent positive as well as the mean fluorescence intensity (MFI) for specific anti-Sm staining are indicated. (B) SK-N-MC cells were seeded in wells of a flat-bottomed 96-well culture plate. After fixation with 1% paraformaldehyde, the wells were reacted with murine monoclonal anti-Sm (5 μg/ml) or human anti-Sm (50 μg/ml) purified from serum IgG fraction of an SLE patient. Murine monoclonal IgG3 (5 μg/ml) or control human IgG (50 μg/ml) of a normal healthy individual was used as controls. Bound IgG was detected with peroxidase-conjugated F(ab’)2 fragments of goat anti-mouse IgG or anti-human IgG. The results are expressed by the absorbance at 492 nm (OD492). Error bars indicate standard deviation (SD) of triplicate determinations.