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. 2022 Jan 31;54(1):472–483. doi: 10.1080/07853890.2022.2031272

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© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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graphic file with name IANN_A_2031272_F0001a_C.jpg — Design of human B cell-focused flow cytometry panel. (A) Sequential gating strategy for identification of B cells and downstream analysis. After lymphocyte gating according to FSC and SSC parameters, CD19-positive lymphocytes were selected and further resolved into distinct subpopulations via the analysis of CD20, CD24, CD27, CD38 or IgD expression. (B) Calculation of absolute numbers. Lymphocytes were gated according to CD45/side scatter dot plots. Then, total B cells were defined as CD3–CD19+ lymphocytes. (C) t-SNE analysis objectively delineates B cell subsets in peripheral blood of healthy individuals. tSNE-based dimension reduction was performed on B cells revealing distinct, shared and diverse lymphocyte phenotypes. Unswitched memory B cells: CD19 + IgD + CD27+ B cells; switched memory B cells: CD19 + IgD–CD27+ B cells; DN B cells: double negative B cells; B10 cells: CD19 + CD24hiCD27+ B cells.

graphic file with name IANN_A_2031272_F0001b_C.jpg — Design of human B cell-focused flow cytometry panel. (A) Sequential gating strategy for identification of B cells and downstream analysis. After lymphocyte gating according to FSC and SSC parameters, CD19-positive lymphocytes were selected and further resolved into distinct subpopulations via the analysis of CD20, CD24, CD27, CD38 or IgD expression. (B) Calculation of absolute numbers. Lymphocytes were gated according to CD45/side scatter dot plots. Then, total B cells were defined as CD3–CD19+ lymphocytes. (C) t-SNE analysis objectively delineates B cell subsets in peripheral blood of healthy individuals. tSNE-based dimension reduction was performed on B cells revealing distinct, shared and diverse lymphocyte phenotypes. Unswitched memory B cells: CD19 + IgD + CD27+ B cells; switched memory B cells: CD19 + IgD–CD27+ B cells; DN B cells: double negative B cells; B10 cells: CD19 + CD24hiCD27+ B cells.