Special Methods Allowed by the Use of the FcRSynd1 Chimera Control of Ligand Clustering

1. CHO cells expressing the FcR-Synd1 chimera, grown in Ham's F-12 medium with 10% fetal calf serum to a confluent monolayer.

2. Ice tray or cold room.

3. Phosphate- or Tris-buffered saline, pH 7.4, at 4°C.

4. Serum-free cell-culture medium at 4°C (Ham's F-12 medium supplemented with 0.2% fatty acid-free bovine serum albumin, Sigma Chemical, St. Louis, MO, cat. no. A-8806).

5. Serum-free cell-culture medium at 37°C (optional).

6. 125I-labeled nonimmune human IgG in lipoprotein buffer: 150 mM NaCl, 0.3 mM Na2EDTA, pH 7.4 at 4°C. Nonimmune human IgG is commercially available (e.g., from Rockland Immunochemicals, Inc., Gilbertsville, PA) and can be radioiodinated with Iodobeads (Pierce Chemical, Rockford, IL).

7. Clustering agent: Unlabeled goat F(ab')2 raised against the Fab domain of human IgG (e.g., from Rockland Immunochemicals).

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