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An optimized protocol to quantify signaling in human transitional B cells by phospho flow cytometry.

Nicholas A Zwang1, Balaji B Ganesh2, Kim T Cardenas3

  • 1Division of Nephrology, Department of Medicine, The University of Illinois at Chicago, 820 South Wood Street (MC 793), Chicago, IL 60612, USA.

Journal of Immunological Methods
|October 16, 2018
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We optimized phospho flow cytometry to analyze signaling in transitional B cells (TrBs). This method allows quantification of signaling in rare B cell subsets, crucial for understanding immune responses and diseases.

Keywords:
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Area of Science:

  • Immunology
  • Cellular Signaling
  • Flow Cytometry

Background:

  • Phospho flow cytometry is vital for analyzing signaling in rare cell populations.
  • Harsh fixation/permeabilization conditions can denature antigens and fluorochromes, hindering analysis of unique B cell subsets.
  • Transitional B cells (TrBs) are an immature subset implicated in transplant rejection, autoimmunity, and malignancy.

Purpose of the Study:

  • To optimize a phospho flow cytometry protocol for quantifying signaling in human transitional B cells (TrBs).
  • To compare signaling in TrBs with mature B cell subsets.

Main Methods:

  • Defined TrBs as CD19+CD24hiCD38hi.
  • Optimized antibody clone selection, epitope labeling sequence, fixation/permeabilization, PMT voltages, and compensation.
  • Developed a protocol for reliable identification of TrBs, naïve, early memory, and late memory B cells.

Main Results:

  • Simultaneously quantified phosphorylation of at least three signaling molecules.
  • Transitional B cells showed diminished MAPK/ERK and Akt phospho signaling upon stimulation.
  • Successfully identified and analyzed signaling in distinct B cell subsets.

Conclusions:

  • An effective protocol was optimized for quantifying B cell subset signaling.
  • This protocol can assess dysfunctional B cell signaling in disease.
  • It may aid in predicting clinical outcomes and monitoring therapies.