Simultaneous Measurement of T-Cell Activation via Proliferation, Cytokine Secretion and Differentiation

Introduction

The biology of T-lymphocyte cells (T-cells) is highly complex and vital to our health and well-being. T-cells come in a number of types, which are defined by specific surface markers such as CD4+ or how they act in response to an immunological stimulus. For exam­ple, cytotoxic T cells (CTLs) kill tumor cells and virally infected cells, whereas regulatory T-cells (T-reg) modulate the activity of CTLs and suppress auto-immune responses.

T-helper cells (Th) can be further specified into subtypes like Th1 and Th17 that secrete different combinations of cytokines once activated by antigen presenting cells. By monitoring changes in cytokine secretion and proliferation this activation can be measured. To properly understand such a complex biology requires a platform capable of multiplexing cell and bead-based readouts using suspension cells.

The Intellicyt® iQue Screener platform is perfectly suited to high content multiparametric screening assays such as the concurrent measurement of T-cell activation through proliferation, cytokine secretion and differentiation.

  • QBeads are compatible with IPT antibodies and with MultiCyt proliferation dye.
  • Measure secreted proteins (cytokines) and proliferation of immunophenotyped (IPT) PBMCs.
  • The ForeCyt® software expertly manages data from complex experiments.

Cytokine-capturing QBeads are clearly separated from PBMCs by their forward and side scatter (left plot). Individual QBeads populations are separated by dyes internal to the beads (upper right) while the cell populations are separated by anti-CD3 and anti-CD8 immunophenotyping antibodies conjugated with FITC and PE, respectively (lower right).Figure 1. Cytokine-capturing QBeads are clearly separated from PBMCs by their forward and side scatter (left plot). Individual QBeads populations are separated by dyes internal to the beads (upper right) while the cell populations are separated by anti-CD3 and anti-CD8 immunophenotyping antibodies conjugated with FITC and PE, respectively (lower right).

Results

By treating PBMCs with PHA, activation is induced which results in proliferation, secretion of pro-inflammatory cytokines and changes in T-cell populations. The Intellicyt® iQue Screener can measure each of these responses in the same well and at the same time. In this example, degree of proliferation of three immunophenotypes, as well as three cytokines was analyzed, placing the results in a highly correlated dataset.

Detection of cytokines by QBeads in the same well in which cells were immunophenotyped. Secreted proteins were detected 2 days after PHA treatment. Data is average +/- S.D. of 3 wells. Secreted profiles differ by dose and by time.

Figure 2. Detection of cytokines by QBeads in the same well in which cells were immunophenotyped. Secreted proteins were detected 2 days after PHA treatment. Data is average +/- S.D. of 3 wells. Secreted profiles differ by dose and by time.

Activation of PBMCs over 2 days resulted in proliferation of T-cells. A) T-cells expressing CD3/FITC and CD8/PE (or neither) were gated in the ForeCyt<sup/>®</sup> software. B) The proliferation of the 3 T-cell populations in response to various PHA doses were found to differ. Data is average +/- S.D. of 3 wells.

Figure 3. Activation of PBMCs over 2 days resulted in proliferation of T-cells. A) T-cells expressing CD3/FITC and CD8/PE (or neither) were gated in the ForeCyt® software. B) The proliferation of the 3 T-cell populations in response to various PHA doses were found to differ. Data is average +/- S.D. of 3 wells.

Summary

  • The Intellicyt® iQue Screener measures multiple parameters from samples containing both beads and cells. In this example, three distinct assays – prolif­eration, cytokine secretion, and IPT were run together and analyzed in a single read. This does not require washing or dividing the assay into multiple reads. It takes just one well, one read, and 6 end points for a highly correlated dataset.
  • The Intellicyt® iQue Screener enables large screening of complex biology with its high throughput. Complexity of the assay is not a factor in how fast the Intellicyt® iQue Screener screens. One 384-well plate containing this complex assay was read in 18 minutes.
  • ForeCyt® software enables rapid management, analysis and visualization of data from complex biology screens. This is performed in real time and allows templating of both gating, analysis and reporting.

IntelliCyt CorporationIntelliCyt Corporation

Intellicyt, A Sartorius brand is the market leader in suspension-cell analysis. The Intellicyt® iQue Screener PLUS platform is a high throughput, suspension cell analysis platform that enables researchers to rapidly profile cell phenotype and function in biologics, small molecule, and cell therapy discovery workflows.

Based on flow cytometry but built for screening, the system allows researchers to automatically acquire and analyze high content, multiplexed assays needed to assess immune cell function by combining cell immuno-phenotyping, cell health, and secreted protein (cytokine) analysis in every well of microtiter plates.


Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.

Last updated: Apr 23, 2019 at 11:16 AM

Citations

Please use one of the following formats to cite this article in your essay, paper or report:

  • APA

    IntelliCyt Corporation. (2019, April 23). Simultaneous Measurement of T-Cell Activation via Proliferation, Cytokine Secretion and Differentiation. News-Medical. Retrieved on May 24, 2019 from https://www.news-medical.net/whitepaper/20190423/Simultaneous-Measurement-of-T-Cell-Activation-via-Proliferation-Cytokine-Secretion-and-Differentiation.aspx.

  • MLA

    IntelliCyt Corporation. "Simultaneous Measurement of T-Cell Activation via Proliferation, Cytokine Secretion and Differentiation". News-Medical. 24 May 2019. <https://www.news-medical.net/whitepaper/20190423/Simultaneous-Measurement-of-T-Cell-Activation-via-Proliferation-Cytokine-Secretion-and-Differentiation.aspx>.

  • Chicago

    IntelliCyt Corporation. "Simultaneous Measurement of T-Cell Activation via Proliferation, Cytokine Secretion and Differentiation". News-Medical. https://www.news-medical.net/whitepaper/20190423/Simultaneous-Measurement-of-T-Cell-Activation-via-Proliferation-Cytokine-Secretion-and-Differentiation.aspx. (accessed May 24, 2019).

  • Harvard

    IntelliCyt Corporation. 2019. Simultaneous Measurement of T-Cell Activation via Proliferation, Cytokine Secretion and Differentiation. News-Medical, viewed 24 May 2019, https://www.news-medical.net/whitepaper/20190423/Simultaneous-Measurement-of-T-Cell-Activation-via-Proliferation-Cytokine-Secretion-and-Differentiation.aspx.

Other White Papers by this Supplier