Immunophenotyping and Function using iQue^® Immune Cell-Based Kits

The iQue^® Human T Cell Activation Cell and Cytokine Profiling Kit assesses the activation status of T cells while providing information about their health and their role in cytokine secretion. This one wash assay requires minimal hands on time and measures both cells and beads to provide the following biological insights:

• Live immune cells are distinguished from dead cells by staining with a fluorescent membrane integrity dye.
• Cells are immunophenotyped by staining with a fluorescent antibody panel to separate CD3+ T cells, CD3- non-T cells, CD4+ T helper cells, CD8+ T cytotoxic cells.
• The T cell phenotypes are profiled for the expression of 3 activation markers, CD69 (early), CD25 (late), and HLA-DR (even later, with strong stimulation).
• Optimized workflow enables the measurement of effector cytokines secreted by activated T cells, Th1 cytokine IFNγ/ multifunctional cytokine TNFα in the same assay well.

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Key Benefits

• Speed: Sample to actionable data by allowing multiple assays to be run in one assay reducing the hands on time, numbers of overall tests and conserving precious sample.
• Results: 15 endpoints from a single well assay measuring T cell phenotypes, early and late cell activation markers in each phenotype, cell viability, cell count, proliferation and secreted effector cytokines.
• Biological insight: Generate cell health and functional data from same cell by multiplexing cell and cytokine detection in one well.
• Simplicity: Single platform assay and data acquisition eliminates the time of acquiring and correlating data from multiple sources.
• Automation friendly: Compatible for automated high-throughput screening with excellent day-to-day reproducibility.

Workflow

The iQue^® Human T Cell Companion Kits can be used to measure additional cytokines besides those already included in the iQue^® Human T Cell Activation Cell and Cytokine Profiling Kit or other T Cell Immunology Kits. Below is the list of available cytokines for T Cell Companion Kits.

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The iQue^® Human T Cell Memory Kit measures T cell memory phenotype and function at different stages while providing information about their health and their role in cytokine secretion. This one wash assay requires minimal hands on time and measures both cells and beads to provide the following biological insights:

• Live immune cells are distinguished from dead cells by staining with a fluorescent membrane integrity dye.
• Cells are immunophenotyped by staining with a fluorescent antibody panel to separate CD3+ T cells, CD3- non-T cells, CD4+ T helper cells, CD8+ T cytotoxic cells.
• The panel also includes 5 different T cell surface markers for T naive/memory/effector cell phenotyping: CD45RA, CD45RO, CD27 (long term survival), CD62L, and CD95.
• Optimized workflow enables the measurement of effector cytokines secreted by activated T cells, including pro-inflammatory cytokine IFNγ and anti-inflammatory cytokine IL-10 in the same assay well.

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Key Benefits

• Speed: Sample to actionable data by allowing multiple assays to be run in one assay reducing the hands on time, numbers of overall tests and conserving precious sample.
• Results: 11 endpoints from a single well assay measuring T cell memory phenotype and function at different stages, cell viability, cell count, and secreted pro/anti-inflammatory cytokines.
• Biological insight: Generate cell health and functional data from same cell by multiplexing cell and cytokine detection in one well.
• Simplicity: Single platform assay and data acquisition eliminates the time of acquiring and correlating data from multiple sources.

Workflow

The iQue^® Human T Cell Killing Kit (CD3, CD4, and CD8) is designed for reliable identification of human T cell subsets. This assay is optimized to run on the iQue^® 3 High-Throughput Screening (HTS) by Cytometry Platform (VBR and VYB configurations), combining high throughput sampling, flow cytometry detection, and multiplexing capabilities.

• T cells are immunophenotyped by staining with a fluorescent antibody panel to separate CD3+, CD4+, and CD8+ cells.
• Compatible with Qbead Panels enabling cytokine detection and cell phenotyping at the same time.
• Optimized workflow provides flexibility by enabling additional cytokines to be added for further characterization of subpopulations.

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Key Benefits

• High-throughput: Designed for high-throughput immunophenotyping of T cells in 96- and 384-well plate format using 10µL (96-well format) and 5µL (384-well format) of cells.
• Biological insights: Generate functional data and option to analyze cell health from same cell by multiplexing cell phenotypes and function in a single well.
• Flexibility: Choose additional cytokines to be added for further characterization of subpopulations.

Workflow

The iQue^® Human T Cell Exhaustion Kit is designed for ease of use in multiplexing markers of T cell exhaustion, phenotyping T helper and T cytotoxic cells, and bead-based measurement of secreted cytokines, all in the same assay. This optimized assay offers these unique advantages:

• Live immune cells are distinguished from dead or membrane compromised cells by staining with a fluorescent membrane integrity dye.
• Cells are immunophenotyped by staining with a fluorescent antibody panel to distinguish CD3+ T cells, CD3- non-T cells, CD3+CD4+ helper T cells, and CD3+CD8+ cytotoxic T cells.
• The panel also includes three markers of T cell exhaustion: PD-1, Tim-3, and Lag-3.
• Optimized workflow enables the measurement of cytokines secreted by activated T cells (IFNγ and TNF) in the same assay well.
• The capacity of the sample cells to proliferate can also be determined with an optional proliferation dye if desired (included with the kit).

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Key Benefits

• Speed: Sample to actionable data by allowing multiple assays to be run in one assay thereby reducing the hands on time, numbers of overall tests and conserving precious sample.
• Results: 11 endpoints from a single-well assay measuring T cell exhaustion phenotypes and function, as well as cytokine secretion in response to stimulation, cell viability, and an optional proliferation dye allows screening for molecules that reverse T cell exhaustion.
• Biological insight: Generate functional data and analyze cell health from the same well by multiplexing cell and cytokine detection in one assay.
• Simplicity: Single platform assay and data acquisition and analysis eliminates the time of acquiring and correlating data from multiple sources.

Workflow

The iQue^® Human NK Cell Killing Kit was designed for ease of use in multiplexing cellular phenotypic and functional markers and bead-based, secreted cytokine measurements in the same assay. This one wash assay requires minimal hands on time and measures both cells and beads to provide the following biological insights:

• Live immune cells are distinguished from dead cells by staining with fluorescent membrane integrity dye.
• Target cells are distinguished from effector cells by staining with a fluorescent encoder dye.
• NK cells are immunophenotyped by staining with a fluorescent antibody panel to separate CD3- CD56+ NK cells. The expression of the IgG Fc receptor III (CD16) on the NK cells can then be assessed.
• The panel also includes 2 different NK cell functional activation markers, CD69 and CD25.
• Optimized workflow enables the measurement of pro-inflammatory effector secreted cytokine IFNγ and pro-apoptotic serine protease Granzyme B in the same assay well.

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Key Benefits

• Speed: Sample to actionable data allows multiple assays to be run in one experiment thereby reducing the hands on time, required numbers of overall tests and conserving precious sample.
• Results: Nine endpoints from a single well assay measuring activation markers (CD69 and CD25), cell membrane integrity, cell count, secreted pro-inflammatory cytokine IFNγ, and pro-apoptotic protease Granzyme B.
• Biological insights: Generate functional data and analyze cell health from same cell by multiplexing cell phenotypes and function in a single well.
• Ease of use: Single platform assay and data analysis with iQue Forecyt^® eliminates the time required for acquiring and correlating data from multiple sources.
• Flexibility: Choose additional cytokine measurements from a validated list of iQue^® Human NK Cell Companion Kits.

Workflow

The iQue^® Human T Cell Phenotyping Kit (CD3, CD4 and CD8) is designed for reliable identification of human T cell subsets. This assay is optimized to run on the iQue^® 3 High-Throughput Screening (HTS) by Cytometry Platform (VBR and VYB configurations), combining high throughput sampling, flow cytometry detection and multiplexing capabilities.

• T cells are immunophenotyped by staining with a fluorescent antibody panel to separate CD3+, CD4+ and CD8+ cells.
• Compatible with Qbead Panels enabling cytokine detection and cell phenotyping at the same time.
• Optimized workflow provides the flexibility by enabling additional markers and cytokines to be added for further characterization of subpopulations.

Request a Quote

Key Benefits

• High-throughput: Designed for high-throughput immunophenotyping of T cells in 96- and 384-well plate format using 10µL (96-well format) and 5µL (384-well format) of cells.
• Biological insights: Generate functional data and option to analyze cell health from same cell by multiplexing cell phenotypes and function in a single well.
• Flexibility: Choose additional markers and cytokines to be added for further characterization of subpopulations.

Workflow

Data Analysis

Phenotyping analysis of human PBMCs cultured for one day. A. Set gate for live cells in all events to exclude debris/dead cells. B. Remove doublets from live cells to obtain single cells. C. CD3+ cells of Singlets. D. CD4+ and CD8+ subsets of CD3+ cells.