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. 2025 Sep 12;16:1632302. doi: 10.3389/fimmu.2025.1632302

Figure 2.

(A) Line graph showing fluorescent area over 25 hours for different antibodies, with FabFluor and Bococizumab showing the highest fluorescence. (B) Scatter plot displaying normalized internalization for Herceptin variants and other antibodies across four donors. (C) Line graph depicting fluorescent area over time, with Bococizumab showing the highest value among listed antibodies. (D) Scatter plot of normalized internalization for different antibodies across four donors, with Herceptin IgG1 and Bococizumab showing varying internalization levels.

FabFluor-pH Orange indirect labeling assay shows distinct internalization profiles of antibody isotypes and Fc variants. The internalization of herceptin variants and IgG4 molecules into DCs were evaluated using IncuCyte® by indirect labeling with FabFluor-pH Orange. (A, C) representative time-course internalization of (A) five herceptin variants and (C) four IgG4 molecules from a single donor. Data points are averages of duplicate wells. (B, D) Endpoint internalization after 24 hours for (B) five herceptin variants and (D) four IgG4 molecules. Data were acquired from four independent donors across multiple days, normalized as described in the Materials and Methods.