Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets

Marsman, Casper; Jorritsma, Tineke; ten Brinke, Anja; van Ham, S. Marieke

doi:10.3390/cells9122633

Submit to this Journal Review for this Journal Edit a Special Issue

Article Menu

...

Open AccessArticle

Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets

¹,

¹ and

^1,2,*

Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, 1066 CX Amsterdam, The Netherlands

Swammerdam Institute for Life Sciences, University of Amsterdam, 1098 XH Amsterdam, The Netherlands

Author to whom correspondence should be addressed.

Cells 2020, 9(12), 2633; https://doi.org/10.3390/cells9122633

Received: 31 July 2020 / Revised: 28 October 2020 / Accepted: 4 December 2020 / Published: 8 December 2020

(This article belongs to the Special Issue B Cell Signaling and Activation in Autoimmunity)

View Full-Text Download PDF

Browse Figures

Review Reports Citation Export

Abstract

The flow cytometric detection of intracellular (IC) signaling proteins and transcription factors (TFs) will help to elucidate the regulation of B cell survival, proliferation and differentiation. However, the simultaneous detection of signaling proteins or TFs with membrane markers (MMs) can be challenging, as the required fixation and permeabilization procedures can affect the functionality of conjugated antibodies. Here, a phosphoflow method is presented for the detection of activated NF-κB p65 and phosphorylated STAT1, STAT3, STAT5 and STAT6, together with the B cell differentiation MMs CD19, CD27 and CD38. Additionally, a TF-flow method is presented that allows the detection of the B cell TFs PAX5, c-MYC, BCL6 and AID and antibody-secreting cell (ASC) TFs BLIMP1 and XBP-1s, together with MMs. Applying these methods on in vitro-induced human B cell differentiation cultures showed significantly different steady-state levels, and responses to stimulation, of phosphorylated signaling proteins in CD27-expressing B cell and ASC populations. The TF-flow protocol and Uniform Manifold Approximation and Projection (UMAP) analysis revealed heterogeneity in TF expression within stimulated CD27- or CD38-expressing B cell subsets. The methods presented here allow for the sensitive analysis of STAT, NF-κB p65 signaling and TFs, together with B cell differentiation MMs, at single-cell resolution. This will aid the further investigation of B cell responses in both health and disease. View Full-Text

Keywords: differentiation; germinal center; antibody-secreting cells; phosphorylated STATs; NF-κB1

▼ Show Figures

Graphical abstract

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

Supplementary Material

Supplementary File 1:
RAR-Document (RAR, 6067 KiB)

Share and Cite

MDPI and ACS Style

Marsman, C.; Jorritsma, T.; ten Brinke, A.; van Ham, S.M. Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets. Cells 2020, 9, 2633. https://doi.org/10.3390/cells9122633

AMA Style

Marsman C, Jorritsma T, ten Brinke A, van Ham SM. Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets. Cells. 2020; 9(12):2633. https://doi.org/10.3390/cells9122633

Chicago/Turabian Style

Marsman, Casper, Tineke Jorritsma, Anja ten Brinke, and S. M. van Ham. 2020. "Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets" Cells 9, no. 12: 2633. https://doi.org/10.3390/cells9122633

Find Other Styles

Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Metrics

Abstract views Pdf views Html views

Article Menu

Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets

Abstract

Supplementary Material

Share and Cite

Article Metrics

Article Access Statistics

Article Access Map by Country/Region

Further Information

Guidelines

MDPI Initiatives

Follow MDPI