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High-parameter T-cell spectral phospho-flow-cytometry.

Gvantsa Pantsulaia1, Joshua Brody1

  • 1Division of Hematology and Medical Oncology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, United States; Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States; Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States.

Methods in Cell Biology
|January 31, 2026
PubMed
Summary

Phospho-flow cytometry, combined with fluorescent cell barcoding, offers robust immunological signaling analysis. This study presents a standardized method for stimulating T cells, improving experimental reproducibility and reducing variability in flow cytometry assays.

Keywords:
Cancer immunotherapyCell signalingFluorescent cell barcodingImmune cell profilingPhospho-flow cytometrySignal transductionT-cell activation

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Area of Science:

  • Immunology
  • Cellular Signaling
  • Flow Cytometry

Background:

  • Phospho-flow cytometry is crucial for studying immunological signaling, enabling simultaneous detection of cell markers and intracellular phospho-proteins.
  • It has potential applications in diagnostics, therapeutics, drug screening, and efficacy assessment.
  • Fluorescent cell barcoding (FCB) enhances flow cytometry throughput and experiment robustness.

Purpose of the Study:

  • To describe a standardized experimental method for stimulating mouse and human T cells.
  • To address inter-operator variability in phospho-flow assays.
  • To provide a adaptable protocol for robust T cell activation.

Main Methods:

  • Development of a standardized T cell stimulation protocol.
  • Integration of phospho-flow with fluorescent cell barcoding (FCB) for multiplexing.
  • Application to mouse and human T cell activation studies.

Main Results:

  • Demonstration of robust T cell activation using the described method.
  • Improved experimental consistency and reduced variability in flow cytometry staining.
  • Successful adaptation for diverse experimental designs.

Conclusions:

  • The presented method provides a standardized approach to T cell stimulation for phospho-flow analysis.
  • This standardization enhances reproducibility and reduces variability in immunological signaling studies.
  • The adaptable protocol supports high-throughput flow cytometry applications.