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. 2019 Jul 17;10:3137. doi: 10.1038/s41467-019-10948-w

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© The Author(s) 2019

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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Fig. 4 — Detection of CD20-positive myeloma cells by flow cytometry and dSTORM and elimination by CD20 CAR-T. a Flow cytometric analysis of CD20 expression on primary myeloma cells purified from bone marrow aspirates. Gating strategy: FSC/SSC plasma cell gate → 7-AAD⁻ → CD138⁺/CD38⁺. Patients with myeloma cells comprising a CD20-positive fraction by FC (patient M025 & patient M027), or being CD20-negative (patient M029) are shown. Data for all patients are shown in Table 3. b Detection of CD20 using dSTORM. Myeloma cells were identified by transmitted light microscopy and expression of CD138 and CD38 as detected by conventional wide-field fluorescence microscopy. CD20 was detected on primary myeloma cells using conventional wide-field fluorescence and dSTORM. Images depict CD20 molecules in the bottom plasma membrane (attached to glass surface) of a CD20-positive (top row) and a CD20⁻ myeloma cell (bottom row). Small panels display magnification of boxed regions revealing the markedly enhanced sensitivity of dSTORM. c Merged expression profiles for CD20 on myeloma cells from patients M025, M027, & M029 generated by dSTORM. Far left and left column: relative CD20 density obtained after staining with isotype control (far left column) and anti-CD20 antibody (left column). Densities are provided as logarithmic numbers (natural logarithm, Ln) of molecules per µm². Density plots were divided into CD20-positive and CD20-negative subpopulations, defined by the density distribution pattern of the isotype control antibody. Density plots were fitted with a one or two-component log-normal function. Right and far right column: relative CD20 density obtained after staining with anti-CD20 antibody on myeloma cells after treatment with CD20 CAR-T (right column) or untransduced control T cells (far right column). Dotted blue line: function of the isotype control. Data for single patients are shown in Supplemementary Table 3 and Supplementary Fig. 8. d Exemplary images of CD20-positive cells as detected by dSTORM. Myeloma cells were identified as stated in b and CD19 was detected on primary myeloma cells using dSTORM for patients M025, M027, and M029. The representative images depict CD20 molecules in the bottom plasma membrane (attached to glass surface). Scale bars, 1 µm and 0.2 µm (magnifications) (b), 3 µm (d)