quantumtm alexa fluor® 647 mesf microsphere kit  (Bangs Laboratories)

Journal: iScience

Article Title: Ligand preference of EphB2 receptor is selectively regulated by N-glycosylation

doi: 10.1016/j.isci.2025.112386

Figure Lengend Snippet: The glycosylation of EphB2 is indispensable for ephrin-A5/B2 binding specificity (A) Top: western blot and Coomassie brilliant blue staining results of purified EphB2 protein before/after PNGase F treatment. Bottom: western blot results of endogenous EphB2 from adult and P10 mice hippocampus before/after PNGase F enzyme treatment. GAPDH served as a loading control. (B) Flow diagram of FCM assay. EphB2-expressing cells bound by pre-clustered ephrin-A5 or ephrin-B2 were recorded by flow cytometry and translated to the relative MESF values. (C) The FCM results are analyzed by the “one-site specific binding” model to fit the saturation binding curves and obtain the K D values. The K D of EphB2 WT to ephrin-B2 and ephrin-A5 are 3.526 nM and 111.9 nM; the K D of EphB2 4Q to ephrin-B2 and ephrin-A5 are 29.91 nM and 32.29 nM. (D and E) Representative confocal microscopy images of ligand-induced ligand/receptor colocalization assays upon stimulating with ephrin ligands. Green represents cells expressing EphB2 4Q, and red represents 647-labeled pre-clustered ephrin ligands. Scale bar: 10 μm. Averaged ratios of cells with EphB2 4Q/ephrins colocalization after stimulation are plotted as (E). Statistical significance was performed by two-way ANOVA; Tukey’s post-hoc multiple comparisons test; ns, not significant; ( n = 3). (F) Representative western blot results of immunoprecipitated C-terminal 3 × FLAG EphB2 WT or EphB2 4Q in HEK293T cells after ephrin-A5 or ephrin-B2 stimulation. (G) Measurement of relative EphB2 tyrosine phosphorylation, correlated with (F). Statistical significance was performed by one-way ANOVA; Tukey’s post-hoc multiple comparisons test, ns, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ( n = 3). All data are represented as mean ± SD.

Article Snippet: Quantumtm MESF microsphere kits , Bangs Laboratories , PSD818.

Techniques: Glycoproteomics, Binding Assay, Western Blot, Staining, Purification, Control, Expressing, Flow Cytometry, Confocal Microscopy, Labeling, Immunoprecipitation, Phospho-proteomics

Journal: Haematologica

Article Title: The interleukin-3 receptor CD123 targeted SL-401 mediates potent cytotoxic activity against CD34 + CD123 + cells from acute myeloid leukemia/myelodysplastic syndrome patients and healthy donors

doi: 10.3324/haematol.2018.188193

Figure Lengend Snippet: Low levels of CD123 are sufficient for SL-401-mediated cytotoxicity. (A) Correlation between CD123 levels and SL-401 induced cytotoxicity in patient AML cells. CD123-MESF on vehicle-treated AML samples (N=13) and viability after 48 hr of SL-401 (1 μg/ml) treatment were used for correlation analysis. (B) Changes in CD123 molecules in AML blasts after SL-401 treatment. CD123-MESF was determined on viable AML blasts after 24 hours of treatment with vehicle or SL-401 (N=13). Mean difference = 11086 (95% CI for mean difference: 2006, 20166), P =0.021. (C-D) CD123 expression and effect of SL-401 on umbilical cord blood derived CD34 + cells. (C) CD34 positive selected cells were used for multicolor flow cytometry analysis and colony formation assays. Colonies were counted 10–14 days after plating CD34 + cells with continuous presence of vehicle or SL-401(1 μg/ml) P =0.019. (D) Effect of SL-401 on umbilical cord blood liquid cultures. Non-enriched Cord blood samples (N=4 CB) were ficoll processed to obtain mononuclear cells and cultured in RPMI media with 20% FBS and GM-CSF, SCF and IL-3 (10 ng/ml) in presence of SL-401 and the cells were counted and immunophenotyped after 48 hours. Live CD34 + cell counts per ml of culture are shown in the graph. Vehicle vs . SL-401 10ng/ml not significant; Vehicle vs . SL-401 100ng/ml; P =0.0356 and Vehicle vs . SL-401 1 μg/ml; P =0.0089.

Article Snippet: CD123 Molecules of Equivalent Soluble Fluorochrome (MESF) on AML cells was calculated using QuantumTM MESF microsphere kit (Bangs Laboratories, Fishers, IN) and CD123 antibody (Clone: SSDCLY107D2; Beckman Coulter) after establishing calibration curve for each experiment per the manufacturer’s instructions.

Techniques: Expressing, Derivative Assay, Flow Cytometry, Cell Culture