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. Author manuscript; available in PMC: 2020 Dec 29.
Published in final edited form as: Nat Nanotechnol. 2020 Jun 29;15(8):716–723. doi: 10.1038/s41565-020-0719-0

Fig. 5. Confocal microscopic imaging of DNA-nanoparticles on Ramos B cells.

Fig. 5.

(a) Time series imaging of Ico-30x, 6HB-2x-28nm, and 6HB-2x-7nm shows surface binding and internalization into Ramos B-cells. Here, eOD-GT8 was fluorescently labelled with Alexa Fluor 647, VRC01 IgM-BCR was labeled with a FAb fragment conjugated to Janelia Fluor 549 prior to antigen addition, and actin was labeled with phalloidin Alexa Fluor 405 after cell fixation. Scale bar is 5 μm. This experiment has been performed twice (n=2 biological replicates) with similar results. (b) Total intensity of eOD-GT8 is highly correlated with intensity of IgM-BCR, confirming specific binding of NPs to the IgM-BCR and co-internalization. Numbers of cells analyzed are 15 (1 min), 23 (5 min), 16 (30 min), cells are from the same culture. (c) Ramos cells were labeled with an anti-phospho-Syk antibody after fixation and the total pSyk intensity per cell was determined. Numbers of cells analyzed are 11 (control), 58 (6HB-2x-7nm), 43 (6HB-2x-28nm), 56 (Ico-30x), and 56 (eOD-GT8–60mer), cells are from the same culture. (d) Internalized fraction of eOD was estimated by segmenting the cell surface using a phalloidin stain as detailed in Methods. Total internal eOD fluorescence was divided by total cellular eOD fluorescence on a cell-by-cell basis. Numbers of cells analyzed are 19 (6HB-2x-7nm), 23 (6HB-2x-28nm), and 15 (Ico-30x), cells are from the same culture. In (c) in (d), error bars denote the standard error of the mean fluorescence between cells with significance determined by a two-sided Student’s t-test (*: p=0.0314; **: p=0.0016;; NS: Not statistically significant, p= 0.3038 for 6HB-2x-28nm/Ico-30x, and p= 0.4153 for 6HB-2x-28nm/eOD-GT8–60mer). In (d), error bars denote the standard error of the mean fluorescence between cells with significance determined by Student’s t-test (*: p=0.0142; **: p=0.0074; NS: Not statistically significant, p=0.55). (All P-values are available in the Supplementary Table 9)