T Cell Activation

The activation of naive T cells by an antigen and costimulatory signals initiates clonal expansion of both CD4+ helper and CD8+ cytotoxic T cells. In addition to T cell proliferation, a variety of signalling pathways are activated, leading to the expression of functional cell surface markers and the release of cytokines.

There is a growing market of therapies (bispecific, checkpoint inhibitor antibodies & CAR-T cells) that focus on using the immune system to target cancer. The ability to monitor and quantify activation profiles allows for a greater understanding of how T cells react to certain stimuli which in turn can provide insight into how well drugs will be able to harness the power of the immune system.

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Assay Concept

Multiplexed approach to measure T cell activation, proliferation and cytokine release in a single well.

Figure 1. Illustration of Human T Cell Activation Kit assay principles. Different T cell phenotypes are profiled for the expression of 3 activation markers: CD69 (early), CD25 (late), and HLA-DR (even later). The 2 effector cytokines (IFNγ and TNFα) are also quantified using 2-plex Qbeads in a sandwich immunoassay format in the same well. Simultaneous measurement of T cell proliferation or encoded target cells is possible, but is not included in this illustration.

Key Advantages

Gain additional biological insight into T cell activation status in physiologically relevant models

Figure 2. Kinase inhibitor screen highlights variation in human T cell activation profiles.

PBMCs were treated with a panel of Kinase inhibitors (20 µM) for one hour. CD3/CD28 Dynabeads were then added to stimulate activation for 24 hours. 10 µL samples were analysed using the T Cell Activation Cell and Cytokine Profiling Kit and the iQue system.

(A) Plate view of CD4+CD69+ gating strategy. Green box identifies media only control. Red box identifies cyclosporine control. Yellow boxes identify the compounds shown on graphs B-D (1: Ruxolitinib; 2: Bisindolylmaleimide I; 3: AZD 7762).

(B, C) Expression of CD69, CD25 and HLA-DR on CD4+ and CD8+ cells.

(D) Release of IFNγ and TNFα cytokines from PBMCs.

Simultaneously quantify surface protein & cytokine expression

Figure 3. Quantify variability in activation profile and cytokine release from PBMCs isolated from different donors.

SKOV-3 cells were seeded (4K/well) in a co-culture assay with PBMCs (10K/well) from 3 donors. Activation was induced by CD3/CD28 Dynabeads. Cells were lifted following the adherent cell lifting protocol after 4 days and analyzed using the T Cell Activation Cell and Cytokine Profiling Kit. (A-C) Expression of markers of early (CD69), late (CD25) and even later (HLA-DR) T cell activation. (D) IFNγ release by each donor.

Easy to use and interpret; pre-defined gating strategy

Figure 4. Pre-defined gates on iQue Forecyt® enable automatic phenotyping of human T cell subsets.

Minimal sample manipulation: low volume, one wash, no dilution

  • Simple protocol where cells and supernatant are taken as one sample allowing for a streamlined workflow.
  • No labelling, or compensation optimization steps required.

Figure 5a. Easy to follow protocol for the analysis of T cell phenotypes and cytokine release using the T Cell Activation Kit.

Figure 5b. Easy to follow modified protocol to allow the inclusion of target cells in the assay. Allowing the analysis of T cell phenotypes and cytokine release using the T Cell Activation Kit.

Ordering Information

T Cell Activation Kit


Compatible with iQue3 VBR configuration

Available Sizes

Catalog Numbers

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1 x 96 well


T Cell Activation Kit

5 x 96 wells


1 x 384 wells


5 x 384 wells


Technical Resources

T Cell Activation Kit Product Guide

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Adherent Cell Lifting Protocol

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A Kinase inhibitor phenotypic screen using a multiplex T cell...

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Application Note

Combined Live Cell and Flow Cytometry Analysis of Immune Cell Killing

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iQue® Human T Cell Activation Kit Protocol Optimization for...

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