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. 2020 Jan 24;10:1130. doi: 10.1038/s41598-020-58002-w

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© The Author(s) 2020

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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Flow cytometric sorting of VSD4-NavAb-specific hybridoma. Shown is the gating strategy used to single cell sort antigen-specific hybridoma from a hybridoma pool generated from mice immunized with VSD4-NavAb proteoliposomes. (A) Using SSC, FSC and 7-AAD stain to gate live cells, the live hybridoma cells were first bulk sorted based on IgG(+) expression and binding to VSD4-NavAb nanodiscs. (B) Following the bulk sort to enrich for antigen-specific binders, live hybridoma cells were single cell sorted based again on IgG(+) expression and binding to VSD4-NavAb nanodiscs. Alexa Fluor 647-conjugated streptavidin was used for detection of biotinylated VSD4-NavAb. IgG on the hybridoma surface was detected by Alexa Fluor 488-conjugated goat anti-mouse IgG.