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. 2017 Dec 18;7:17748. doi: 10.1038/s41598-017-17669-4

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Confirmation of intracellular localization of PFC. (a) Confocal image (×63) showing perinuclear localization of dual-mode PFC nanoemulsion (in red) in CAR T cells. Nucleus is stained in blue and cell membrane in green via CD3-FITC (b) Control unlabeled CAR T cells do not exhibit red fluorescence. (c) Electron microscopy (EM) micrograph of ultra-thin PFC-labeled CAR T cells reveals the presence of bright vesicles (inset = ×11,000) corresponding to internalized PFC droplets. (d) In control unlabeled cells, no PFC vesicles are found (×4800, scale bars = 1 μm). (e) Flow cytometry scatter plot showing the resulting shift in red fluorescence of CAR T cells after labeling. All cells are labeled after overnight incubation as evidenced by fluorescence gap between labeled (e) and unlabeled (f) CAR T cells. (g) Longitudinal red fluorescence measurement after labeling shows persistent signal and consistent decrease of fluorescence intensity due to cell division.