iQue® Immune Cell-based kits for immunophenotyping and function

iQue^® Immune Cell-Based Kits enable the analysis of several immunological cell phenotypes and functional traits like proliferation, viability and cytokine release. The majority of these kits are improved for low-wash protocols.

Each of these is available in various sizes to fulfill the screening requirements of users. The Forecyt^® software has automated templates for the majority of these kits, thereby making it simple to obtain and examine data. All have been improved for use on the iQue^® Screener platforms.

Available iQue^® phenotype and function kits include:

iQue^® Human T Cell Activation Kit:

Single wash assay offers a multiplexed method to quantify T-cell activation, and proliferation and measure cytokine release in a single well.

iQue^® Human T Cell Companion Kit:

Users can concurrently quantify extra cytokines besides those offered in T Cell Immunology Kit.

iQue^® Human T Cell Memory Kit:

Analyze T cell memory phenotype and function at various stages with the Cytokine Profiling Kit and new T Cell Memory Cell.

iQue^® Human General Immune Cell Killing Kit:

Users can assess cellular phenotypic and functional markers and bead-based, secreted cytokine measurements in a similar assay.

iQue^® Human T Cell Killing Kit:

Assess T cell immunophenotypes and function, cell count, cell health and secrete pro-inflammatory cytokines in a single assay.

iQue^® Mouse T Cell Kit:

Assess T cell activation, memory, exhaustion and cytokine secretion in the same well.

iQue^® Human T Cell Exhaustion Kit:

Availability of phenotyping T helper and T cytotoxic cells, multiplex T cell exhaustion, and bead-based measurement of secreted cytokines, all in the same assay.

iQue® Human NK Cell Killing Kit:

Quantify activation markers, cell count, cell membrane integrity, secreted pro-inflammatory cytokines and pro-apoptotic protease from an individual assay.

iQue^® Human T Cell Phenotyping Kit:

Users can evaluate the phenotype and function of multiple T subsets in the same well.

iQue^® Human T Cell Activation Kit

The iQue^® Human T Cell Activation Cell and Cytokine Profiling Kit evaluate the activation status of T cells while offering data about their health and their role in cytokine secretion. This one wash assay needs the least hands-on time and quantifies both cells and beads to offer the following biological insight:

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

Key benefits

• Speed: Sample to actionable data by enabling multiple assays to be run in a single assay by decreasing the hands-on time, number of overall tests and preserving useful samples.
• Biological insight: It is possible to produce cell health and functional data from the same cell by multiplexing cell and cytokine detection in a single well.
• Results: 15 endpoints obtained from a single well assay quantifying T cell phenotypes, cell viability, early and late cell activation markers in each phenotype, cell count, proliferation, and secreted effector cytokines.
• Automation friendly: Compatible for automated high-throughput screening with outstanding daily reproducibility.
• Simplicity: Single platform assay and data acquisition help remove the time of obtaining and correlating data from several sources.

Workflow

Image Credit: Sartorius UK Ltd

Table 1. Source: Sartorius UK Ltd

iQue^® Human T Cell Companion Kits

The iQue^® Human T Cell Companion Kits could be utilized to quantify extra cytokines besides those already included in the iQue^® Human T Cell Activation Cell and Cytokine Profiling Kit or other T Cell Immunology Kits. Listed below is the list of available cytokines for T Cell Companion Kits.

Table 2. Source: Sartorius UK Ltd

iQue^® Human T Cell Memory Kit

The iQue^® Human T Cell Memory Kit quantifies T cell memory phenotype and function at various stages while offering information about their health and their role in cytokine secretion. This one wash assay requires the least hands-on time and quantifies both cells and beads to offer the following biological insights:

• Live immune cells are differentiated from dead cells through staining with a fluorescent membrane integrity dye.
• Also, the panel consists of five different T cell surface markers for T naïve or memory/effector cell phenotyping: CD45RA, CD45RO, CD27 (long-lasting survival), CD95, and CD62L.
• Improved workflow allows the measurement of effector cytokines secreted by activated T cells. This includes pro-inflammatory cytokine IFNγ and anti-inflammatory cytokine IL-10 in the same assay well.
• Cells are immunophenotyped by staining with a fluorescent antibody panel to isolate CD3+ T cells, CD4+ T helper cells, CD3- non-T cells, and CD8+ T cytotoxic cells.

Key benefits

• Speed: Sample to actionable data by enabling multiple assays to be run in one assay thereby decreasing the hands-on time, number of overall tests performed, and preserving valuable samples.
• Simplicity: Single platform assay and data acquisition remove the time of obtaining and correlating data from various sources.
• Biological insight: Produce cell health and functional data from the same cell by multiplexing cell and cytokine detection in a single well.
• Results: 11 endpoints can be obtained from a single well assay quantifying T cell memory phenotype and function at different stages, cell count, cell viability, and secreted pro or anti-inflammatory cytokines.

Workflow

Image Credit: Sartorius UK Ltd

Table 3. Source: Sartorius UK Ltd

iQue^® Human General Immune Cell Killing Kit

The iQue^® Human General Immune Cell Killing Kit was developed for user-friendliness in multiplexing cellular phenotypic and functional markers and bead-based secreted cytokine measurements in the same assay. This no-wash assay needs the least hands-on time and quantifies both cells and beads to offer the subsequent biological understanding:

• Live immune cells are differentiated from dead cells by staining with fluorescent membrane integrity dye and mitochondrial membrane potential dye.
• Target cells are differentiated from effector cells by staining with a fluorescent encoder dye.
• Concurrent analysis of multiple mechanisms of immune cell-mediated cell killing in a single, high content assay such as cell count, loss of cell membrane integrity, mitochondrial membrane depolarization, and pro-apoptotic protease Granzyme B.

Key benefits

• Speed: Expedient sample to actionable data enables multiple assays to be run in one experiment thereby decreasing the hands-on time, essential numbers of overall tests, and preserving precious samples.
• Biological insights: Analysis of multiple mechanisms of immune cell-mediated cell killing in one well.
• Ease of use: Individual platform assay and data analysis with iQue Forecyt^® removes the time needed for obtaining and correlating data from multiple sources.
• Results: Five endpoints gathered from a single well assay help quantify target and effector cells, cell membrane integrity, mitochondrial membrane depolarization, cell count, and secreted pro-apoptotic protease Granzyme B.

Workflow

Image Credit: Sartorius UK Ltd

Table 4. Source: Sartorius UK Ltd

iQue^® Human T Cell Killing Kit

The iQue^® Human T Cell Killing Kit (CD3, CD4, and CD8) has been specifically developed for the trustworthy identification of human T cell subsets. This assay has been improved to run on the iQue^® 3 (VBR and VYB configurations) platform integrating flow cytometry detection, multiplexing capabilities, and high throughput sampling.

• T cells are immunophenotyped through staining with a fluorescent antibody panel to isolate CD3+, CD4+, and CD8+ cells.
• Improved workflow offers flexibility by allowing extra cytokines to be added for additional characterization of subpopulations.
• Compatible with Qbead Panels allowing cytokine detection and cell phenotyping simultaneously.

Key benefits

• High-throughput: Developed for high-throughput immunophenotyping of T cells in 96- and 384-well plate format utilizing 10 µL (96-well format) and 5 µL (384-well format) of cells.
• Biological insights: Produce functional data and option to examine cell health from the same cell by multiplexing cell phenotypes and function in an individual well.
• Flexibility: Select extra cytokines to be added for additional characterization of subpopulations.

Workflow

Image Credit: Sartorius UK Ltd

Table 5. Source: Sartorius UK Ltd

iQue^® Mouse T Cell Kit

The iQue^® Mouse T Cell Kit quantifies T cell activation, memory and exhaustion while offering data regarding their health and their role in the secretion of cytokine. This one wash assay needs the least hands-on time and quantifies both cells and beads to offer the following biological insights:

• Live immune cells are differentiated from dead cells by staining with a fluorescent membrane integrity dye.
• The panel also consists of four different T cell surface markers for T cell activation, memory and exhaustion in the mouse.
• Cells are immunophenotyped through staining with a fluorescent antibody panel to isolate CD3- non-T cells, CD3+ T cells, CD4+ T helper cells, and CD8+ T cytotoxic cells.
• Improved workflow allows the measurement of effector cytokines secreted by T cells, such as pro-inflammatory cytokine IFNγ and anti-inflammatory cytokine IL-2 in the same assay well.

Key benefits

• Speed: Sample to actionable data by enabling several parameters to be evaluated in one assay decreasing the hands-on time, number of overall tests, and saving precious samples.
• Simplicity: Single platform assay and data acquisition remove the time of obtaining and correlating data gathered from multiple sources.
• Biological insight: Produce cell health and functional data from the same cell by multiplexing cell and cytokine detection in a single well.
• Results: 11 endpoints were obtained from a single well assay quantifying T cell activation, memory, cell viability, exhaustion, cell proliferation and secreted pro- or anti-inflammatory cytokines in the mouse.

Workflow

Image Credit: Sartorius UK Ltd

Table 6. Source: Sartorius UK Ltd

iQue^® Human T Cell Exhaustion Kit

The iQue^® Human T Cell Exhaustion Kit has been specifically developed for user-friendliness in multiplexing markers of phenotyping T helper and T cytotoxic cells, T cell exhaustion, and bead-based measurement of secreted cytokines, all in the same assay. This improved assay provides the benefits outlined below:

• Live immune cells are differentiated from dead or membrane made of cells by staining with a fluorescent membrane integrity dye.
• Cells are immunophenotyped through staining with a fluorescent antibody panel to differentiate CD3+ T cells, CD3- non-T cells, CD3+CD4+ helper T cells, and CD3+CD8+ cytotoxic T cells.
• Also, the panel consists of three markers of T cell exhaustion: PD-1, Tim-3, and Lag-3.
• Improved workflow allows the measurement of cytokines that have been secreted by activated T cells (IFNγ and TNF) in the same assay well.
• The capacity of the sample cells to proliferate can be identified with an optional proliferation dye if preferred (included with the kit).

Key benefits

• Speed: Sample to actionable data by allowing multiple assays to be run in one assay thereby reducing the hands-on time, number of overall tests, and conserving precious samples.
• Biological insight: Produce functional data and examine cell health from the same well through multiplexing cell and cytokine detection in a single assay.
• Simplicity: Single platform assay and data acquisition and analysis remove the time of obtaining and correlating data from several sources.
• Results: 11 endpoints can be obtained from a single-well assay quantifying T cell exhaustion phenotypes and function, as well as cytokine secretion in reaction to cell viability, stimulation, and an optional proliferation dye enables screening for molecules that help in reverse T cell exhaustion.

Workflow

Image Credit: Sartorius UK Ltd

Table 7. Source: Sartorius UK Ltd

iQue^® Human NK Cell Killing Kit

The iQue^® Human NK Cell Killing Kit was specially developed for user-friendliness in multiplexing cellular phenotypic and functional markers and bead-based secreted cytokine measurements in the same assay.

This one wash assay needs the least hands-on time and quantifies both cells and beads to offer the following biological insights listed below

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

Key benefits

• Speed: Sample to actionable data enables several assays to be run in a single experiment which helps decrease hands-on time, the required numbers of overall tests, ultimately saving precious samples.
• Flexibility: Users can select extra cytokine measurements from a validated list of iQue^® Human NK Cell Companion Kits.
• Results: Nine endpoints can be obtained from a single well assay quantifying activation marker (CD69 and CD25), cell count, cell membrane integrity, secreted pro-inflammatory cytokine IFNγ, and pro-apoptotic protease Granzyme B.
• Ease of use: Single platform assay and data analysis with ForeCyt^® removes the time needed for obtaining and correlating data obtained from several sources.
• Biological insights: Generate functional data and examine cell health from the same cell by multiplexing cell phenotypes and function in an individual well.

Workflow

Image Credit: Sartorius UK Ltd

Table 8. Source: Sartorius UK Ltd

iQue^® Human T Cell phenotyping Kit (CD3, CD4, and CD8)

The iQue^® Human T Cell Phenotyping Kit (CD3, CD4, and CD8) has been developed for the trustworthy identification of human T cell subsets. This assay is improved to run on the iQue^® 3 (VBR and VYB configurations) platform integrating high throughput sampling, multiplexing capabilities and flow cytometry detection.

• T cells are immunophenotyped through staining with a fluorescent antibody panel to isolate CD3+, CD4+, and CD8+ cells.
• Optimized workflow offers flexibility by allowing extra markers and cytokines to be added for additional characterization of subpopulations.
• Consistent with Qbead Panels, allowing simultaneous cytokine detection and cell phenotyping.

Key benefits

• High-throughput: Developed for high-throughput immunophenotyping of T cells in 96- and 384-well plate format with the help of 10 µL (96-well format) and 5 µL (384-well format) of cells.
• Flexibility: Users can select extra markers and cytokines to be added for additional characterization of subpopulations.
• Biological insights: Produce functional data with the option to examine cell health from the same cell by multiplexing cell phenotypes and function in an individual well.

Image Credit: Sartorius UK Ltd

Data analysis

Image Credit: Sartorius UK Ltd

Phenotyping analysis of human PBMCs cultured for one day. A. Set gate for live cells into all activities excluding debris or dead cells. B. Eliminate doublets from live cells to achieve single cells. C. CD3+ cells of Singlets. D. CD4+ and CD8+ subsets of CD3+ cells.

Table 9.  Source: Sartorius UK Ltd