Figure 1.

Kinetics of the cell surface IgG binding and ColXVII expression. (A) Immunofluorescence staining for IgG bound to fixed NHEKs. NHEKs were fixed with paraformaldehyde and permeabilized. Cells were incubated with BP IgGs (BP-1, BP-2, or BP-3, 2 mg/ml), normal IgG (2 mg/ml), or rabbit anti-human ColXVII COOH IgG (ColXVII IgG, 12 μg/ml) and then subsequently stained with FITC- or Alexa Fluor 488-conjugated secondary antibodies (green). The nucleus was stained with DAPI (blue). Scale bar 10 μm. (B) Quantification of the fluorescence intensity of IgG bound to fixed NHEKs. Data were analyzed using ICY software. (C) Evaluation of the cell surface IgG binding using flow cytometry. NHEKs were cocultured with 2 mg/ml IgGs for the indicated times and then immediately fixed without permeabilization. The cells were directly incubated with anti-human IgG-FITC and were examined using flow cytometry. (D) Evaluation of the cell surface ColXVII expression using flow cytometry. NHEKs were cocultured with 2 mg/ml IgGs for the indicated times, and then immediately fixed without permeabilization. Then, cells were first incubated with an IgG specific for human ColXVII-COOH, and then incubated with Alexa Fluor 488-conjugated anti-IgG. Cells were examined using flow cytometry. (E) Fluorescence microscopy images of the binding of the indicated IgGs to NHEKs. NHEKs were incubated with BP IgGs (BP-1, BP-2, or BP-3, 2 mg/ml), normal IgG (2 mg/ml), or ColXVII IgG (12 μg/ml) for the indicated times. Then, the cells were fixed, permeabilized, subsequently stained with FITC- or Alexa Fluor 488-conjugated secondary antibodies (green). The nucleus was stained with DAPI (blue). Scale bar 10 μm. (F) Quantification of the fluorescence intensity of IgG bound to NHEKs. Data were analyzed using ICY software.