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. 2001 Mar;45(3):664–672. doi: 10.1128/AAC.45.3.664-672.2001

FIG. 2.

FIG. 2

Binding of sgp120 to CEM-SS cells demonstrated by flow cytometry. CEM-SS (CD4-, CXCR4-, and CCR5-positive) cells were either mock treated or treated with unlabeled anti-Leu3a MAb for 30 min at 4°C and washed twice before addition of sgp120 that was either mock treated or treated with CV-N (molar ratio of sgp120 to CV-N, 1 to 2.4). The sgp120 was added to CEM-SS cells, and the binding was determined from the sgp120 signal using flow cytometry, where anti-gp120 MAb–FITC acquisition indicates overall sgp120 binding, and by anti-Leu3a MAb–PerCP staining, where loss of availability of the Leu3a epitope is an indirect indication of CD4-dependent sgp120 binding. (A) sgp120 signal. CEM-SS cells are sgp120 negative (black trace). Addition of untreated sgp120 results in acquisition of anti-gp120 MAb–FITC staining (compare black and blue traces). CV-N treatment of sgp120 inhibits overall sgp120 binding (compare blue and red traces). Approximately 74% of overall sgp120 binding is CD4 dependent (compare green and blue traces). MFI values for untreated sgp120: binding to untreated cells, 100.3, binding to unlabeled anti-Leu3a MAb-pretreated cells 30.1. CV-N inhibits CD4-dependent and CD4-independent binding of sgp120; note the increased inhibition of sgp120 binding for CV-N-treated sgp120 on unlabeled anti-Leu3a MAb-pretreated cells (compare green and orange traces). (B) Leu3a signal (gp120-binding epitope on CD4). CEM-SS cells express the Leu3a epitope on CD4 (black trace), and saturating unlabeled anti-Leu3a MAb pretreatment blocks binding of anti-Leu3a MAb–PerCP (compare green and black traces). Binding of sgp120 blocks the Leu3a epitope (compare black and blue traces). Cells incubated with CV-N-treated sgp120 have availability of Leu3a epitopes comparable to that of those incubated with untreated sgp120 (compare red and black traces). (C) OKT4 signal (non-gp120-binding epitope on CD4). Neither sgp120 nor CV-N interferes with the level of CD4 or the availability of the OKT4 epitope. At least 10,000 events were acquired for each sample.