Figure 2. Recombinant α-CD99-A192 binds specifically to CD99.

CD99⁺ AML cell lines MOLM-13, MV4-11, U937 cells, and CD99⁻ 293T cells (0.5 x 10⁶cellls) were treated with 1 μM or 10 μM of rhodamine labeled α-CD99-A192 or rhodamine-labeled A192 for 30 minutes on ice. The binding was measured via flow cytometry by analyzing the MFI of rhodamine and normalizing data to untreated cells. The significant binding was observed in A) MOLM-13 B) MV4-11 C) U937 cells. D) No binding was observed in 293T cells. The competitive binding assay was performed in MOLM-13 cells by pre-treating cells with either CD99 Mab (2.5 μg/mL or 5 μg/mL) or IgG and then observing binding with rhodamine α-CD99-A192 (10 μM). The binding was measured via E) Flow cytometry by measuring the peak shift in rhodamine to bound cell and quantifying based on mean fluorescence intensity and F) laser scanning confocal microscopy where cell fluorescence was quantified with ImageJ. Data represented as mean ± SD. * p≤0.05 ** p≤0.01*** p≤0.001**** p≤0.0001