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. 2023 Apr 3;120(15):e2217590120. doi: 10.1073/pnas.2217590120

Fig. 2.

Fig. 2.

IgG3 antibodies are superior to IgG1 antibodies at inducing Fc-mediated phagocytosis further potentiated when used as oligoclonal cocktails. (A) Illustration showing the three different phagocytic states cells can be in and how they are seen in flow cytometric gates: first depicting nonassociated cells, second associated but not internalized, and third with internalized beads. The gate to the right shows a double-positive population signifying internalized beads. Furthest to the right is a graph showing IgG isotype controls. Y axis shows the percentage of THP-1 cells with internalized beads. The same experimental conditions were used as in B and C. (B) IgG1 and IgG3 variants of each antibody clone mediate phagocytosis to varying degrees. The bar graph shows the percentage of cells with internalized beads. 10 µg/mL of the corresponding mAb was used to opsonize the beads (3 million beads per 100,000 phagocytes, a multiplicity of prey 30 (MOP 30). (C) Comparison between IgG1 and IgG3 for each clone with bead signal average for the whole THP-1 population. (D) The graph depicts how monoclonals compare to the cocktails in terms of bead quantity that is being phagocytosed by the whole cell population. The table to the right shows the mean values for the median bead signal (fluorescence intensity) for each treatment. (E) Comparison between IgG1 and IgG3 monoclonal antibodies and cocktails in terms of percentage of THP-1 cells with internalized beads. The table to the right summarizes the data from the graph with mean values from three experiments. In D and E 1.5 million beads per 100,000 phagocytes (MOP 15) were opsonized with 5 µg/mL of either monoclonal antibody or IgG cocktail. Statistical analysis was performed with a two-tailed t test in B and C. For D and E, statistical analysis was done with one-way ANOVA with multiple comparisons corrected with Tukey’s test. P value below 0.05 is denoted by *, P value below 0.01 is denoted by **, and above 0.05 is denoted as ns. Three independent experiments were performed. Bars represent mean values, and error bars represent SEM. Three independent experiments were done for all experiments.