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Figure 11.3. DC-SIGN on DC binds HIV-1 gp120 with high a½nity, in contrast to CD4 and
CCR5. DC express high levels of DC-SIGN and low levels of both CD4 and CCR5 as determined
by ¯ow cytometry (A). The HIV-1 gp120 binding is measured by the ¯uorescent bead gp120 binding
assay on the ¯ow cytometer. Fluorescent beads are indirectly coated with HIV-1 gp120 proteins
(Geijtenbeek et al., 2000a) (B) and added to DC. After 30 min at 37 C, the binding of ¯uorescent
gp120 beads is measured by ¯ow cytometry. The DC are gated based on forward-side-scatter pat-
tern and the fraction of cells binding ¯uorescent beads is measured by ¯ow cytometry (C ). The
speci®city of the gp120 binding is determined by preincubating the DC with blocking antibodies
against CD4 or DC-SIGN, and performing the gp120 binding assay. Blocking antibodies against
DC-SIGN prevent gp120 binding and binding is mediated by the C-type lectin domain of DC-
SIGN since both the polycarbohydrate mannan and the calcium-ion chelator EGTA inhibit the
binding (D).
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