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. 2022 Jan 5;8(1):eabk2855. doi: 10.1126/sciadv.abk2855

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Fig. 1. — (A) Assay overview: Cells genetically encoding a Gal8-mRuby fusion fluorescence protein exhibited diffuse cytosolic mRuby signal in the absence of endosomal disruption. Endosomal disruption caused by NPs carrying Cy5-labeled nucleic acid NPs allows Gal8-mRuby to bind to intra-endosomal glycans, resulting in punctate fluorescent spots. (B) Typical field of view (taken from 80 per NP formulation) imaged by high-throughput fluorescence microscopy of B16-F10 murine melanoma cells after 6-hour exposure to PBAE NPs carrying Cy5-mRNA. Cell identification was done using Hoechst 33342 staining of cell nuclei. Identification of Gal8-mRuby puncta and Cy5 puncta was used to quantify endosomal disruption and NP uptake, respectively. Scale bars, 50 μm. (C) Representative distributions of the Gal8 puncta or Cy5 puncta count per cell obtained from image analysis data.