American Society of Hematology

Fig. 4.

$Fig. 4. Comparison of the abilities of antibodies to redistribute antigens on the cell surface assessed by FRET and Triton X-100 insolubility. / Analysis of CD20 FRET (A and B) and the association of CD20 into membrane rafts (C) in the presence of various anti-CD20 mAbs. (A) Homoassociation analysis of CD20 with B1 and Ritux mAb. Daudi cells were labeled with equimolar mixtures of Cy5 (acceptor)–conjugated mAbs and either unconjugated mAbs (filled histograms) or Cy3 (donor)–conjugated mAbs (open histograms) for 50 minutes at 37°C. Associations were estimated by flow cytometric analysis as described in “Materials and methods.” (B) Comparison of FRET with anti-CD20, anti-MHCII (A9-1), anti-CD37 (WR17), and anti-CD38 (1B4). EHRB and Daudi cells were labeled with equimolar mixtures of Cy3 (donor)– and Cy5 (acceptor)–conjugated mAb pairs for 50 minutes at 4°C (▩) or 37°C (▪) as indicated. Associations were estimated by flow cytometric analysis as described in “Materials and methods.” Data illustrate FRET values ± SEMs, expressed in terms of Cy5 emission at 488 nm, for 3 independent experiments for CD20, MHCII, CD37, and CD38 homoassociation as indicated. (C) Daudi cells were treated with anti-CD20 mAbs (B1, Ritux, 1F5, as indicated) or left untreated (Cont, Ct/Lyn), lysed after 20 minutes in 1% Triton X-100 lysis buffer, and fractionated on a discontinuous sucrose gradient as described in “Materials and methods.” Gradient fractions were resolved on 15% sodium dodecyl sulfate gels, and Western blots developed using 7D1 primary antibody to identify CD20 or rabbit anti-Lyn polyclonal antisera. The pelleted fraction from each sucrose density gradient did not contain CD20 (data not shown).$

Comparison of the abilities of antibodies to redistribute antigens on the cell surface assessed by FRET and Triton X-100 insolubility.

Analysis of CD20 FRET (A and B) and the association of CD20 into membrane rafts (C) in the presence of various anti-CD20 mAbs. (A) Homoassociation analysis of CD20 with B1 and Ritux mAb. Daudi cells were labeled with equimolar mixtures of Cy5 (acceptor)–conjugated mAbs and either unconjugated mAbs (filled histograms) or Cy3 (donor)–conjugated mAbs (open histograms) for 50 minutes at 37°C. Associations were estimated by flow cytometric analysis as described in “Materials and methods.” (B) Comparison of FRET with anti-CD20, anti-MHCII (A9-1), anti-CD37 (WR17), and anti-CD38 (1B4). EHRB and Daudi cells were labeled with equimolar mixtures of Cy3 (donor)– and Cy5 (acceptor)–conjugated mAb pairs for 50 minutes at 4°C (▩) or 37°C (▪) as indicated. Associations were estimated by flow cytometric analysis as described in “Materials and methods.” Data illustrate FRET values ± SEMs, expressed in terms of Cy5 emission at 488 nm, for 3 independent experiments for CD20, MHCII, CD37, and CD38 homoassociation as indicated. (C) Daudi cells were treated with anti-CD20 mAbs (B1, Ritux, 1F5, as indicated) or left untreated (Cont, Ct/Lyn), lysed after 20 minutes in 1% Triton X-100 lysis buffer, and fractionated on a discontinuous sucrose gradient as described in “Materials and methods.” Gradient fractions were resolved on 15% sodium dodecyl sulfate gels, and Western blots developed using 7D1 primary antibody to identify CD20 or rabbit anti-Lyn polyclonal antisera. The pelleted fraction from each sucrose density gradient did not contain CD20 (data not shown).

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