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. 2019 Feb 21;104(9):1841–1852. doi: 10.3324/haematol.2018.207266

Figure 4.

Figure 4.

CD20 and CD37 mAbs colocalize on B cells. (A) Confocal fluorescence microscopy analysis to detect colocalization of cell-bound CD20 and CD37 mAbs. Raji cells were opsonized with hexamerization-enhanced A488-conjugated CD20 mAb 7D8-derived Hx-CD20-7D8 (image 1, green) and hexamerization-enhanced A594-conjugated CD37 mAb 37.3-derived Hx-CD37 (image 2, red), and incubated for 15 minutes (min) at room temperature. Images were captured in PBS imaging medium at ambient temperature using a Zeiss Axi-oObserver LSM 700 microscope with Plan-Apochromat 63X/1.40 Oil DIC M27 objective lenses and acquired/processed using Zen software. Two excitation lasers were used at 488 and 555 nm. In the merged image, overlap of red and green produces orange or yellow. A representative example of two replicate experiments is shown. (B and C) FRET analysis to detect the molecular proximity of (B) WT type I CD20 mAb 7D8 (IgG1-CD20-7D8) or (C) WT type II CD20 mAb 11B8 (IgG1-CD20-11B8), WT CD37 mAb 37.3 (IgG1-CD37) or a combination thereof on the cell membrane of Daudi cells. (D and E) FRET analysis to detect the molecular proximity of hexamerization enhanced variants of (D) type I CD20 mAb 7D8-derived Hx-CD20-7D8 or (E) type II CD20 mAb 11B8-derived Hx-CD20-11B8, CD37 mAb 37.3-derived Hx-CD37 or a combination thereof on the cell membrane of Daudi cells. Daudi cells were opsonized with 10 μg/mL A555-conjugated-and 10 μg/mL A647-conjugated antibody variants for 15 min at 37°C. FRET was calculated from mean fluorescence intensity values as determined by flow cytometry. Data shown are mean and Standard Deviation of six replicates collected from three independent experiments. ****P<0.0001.