FIG E3.

Siglec-8 shuttled to the cell surface can be detected using a sequential labeling step. Eosinophils were labeled with unconjugated anti–Siglec-8 mAb (2C4) and incubated at 37°C to permit endocytosis as in Fig 1. Following detection of remaining anti–Siglec-8 at the cell surface with a FITC-conjugated secondary mAb (‘‘Original’’), Siglec-8 newly expressed at the cell surface (‘‘Shuttled’’) was detected using anti–Siglec-8 (2C4) directly conjugated to Alexa Fluor 647. Control antibodies of the same isotype (iso) are included as negative staining controls. Loss of FITC signal and gain of Alexa Fluor 647 signal at 120 minutes when using anti–Siglec-8 mAbs in both channels represent receptor endocytosis and shuttling to the cell surface. Effective blockade of Alexa Fluor 647–conjugated anti–Siglec-8 binding can be seen at 0 minute when anti–Siglec-8 mAbs are used for both channels (no additional Alexa Fluor 647 signal). FITC, Fluorescein isothiocyanate.