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Optimized flow cytometry assays to detect immune cells
The Invitrogen eBioscience essential phenotyping kits are an efficient and cost-effective flow cytometry assays. These ready-to-use kits have been designed for the reliable identification of T cell subsets including CD4+, CD8+, and regulatory T cell (Tregs) in human blood.
- Instrument agnostic—antibodies, protocol, and gating strategy are compatible with any flow cytometer instrument equipped with violet 405 nm, blue 488 nm, and red 633 nm lasers
- Comprehensive analysis strategy—a standardized data acquisition and analysis plan is provided to perform flow cytometry characterization
- Optimized antibody and reagent concentrations—panels contain the best combinations of antibodies and dyes for maximum sensitivity from a multicolor fluorescence experiment
Need something unique? You can easily incorporate or design a customized panel with our technical specialists.
Workflow
Step 1
- Stimulate cells from peripheral mononuclear cells (PBMCs), lysed whole blood samples, or single-cell suspensions from human tissue for protein expression. This step is only required for eBioscience Essential Human Th1/Th17 Phenotyping Kit.
Step 2
- Use buffer set included in kit for optimal intracellular staining.
- Add LIVE/DEAD reagent to gate out debris and artifacts.
- Add labeled antibodies against cell surface and intracellular markers to enable cell identification and functionality characterization.
Step 3
- Use instrument and compensation beads to set up flow cytometer for optimal cell population determinations.
Click image to enlarge
Step 4
- Set voltages and suggested gating strategies for cell analysis.
Figure 1. Overview of the T cell subset workflow. No extensive panel design is needed. You can just follow the protocol to generate your data. The gating strategy gives guidance to find the correct cell populations when acquiring events. Results will show the percentage of T cells positive or negative for the indicated markers.
Kits for T cell immunophenotyping
Validated antibody flow panels
T cell
CD3:APC-eF780
CD4:APC
CD8:eF450
CD62L:FITC
CCR7:PE
Treg
CD4:FITC
CD25:PerCP-eF710
CD127:PE
Foxp3:eF450
Th1/Th17
CD4:FITC
IL-17:APC
IFN-γ:PE
Figure 2. T cells can be categorized into subset populations based on the particular immune response(s) they mediate. CD8+ T cells directly kill infected or tumor cells via perforin and granzymes. CD4+ cells regulate other immune cells through adaptive immunity responses. CD4+ cells can be stimulated to differentiate into multiple populations: for example, resulting Th1 cells influence macrophage and B cells and resulting Th17 cells secret cytokines and other factors for an inflammatory response. The Treg population inhibits or downregulates the immune response.
Instrument specifications required for analyzing eBioscience phenotyping kits
To run panels within these kits, instruments are required to have a 2–3 laser system with flexible filter configurations. Below is an example instrument configuration achievable for most traditional flow cytometers.
| Instrument | Specification |
|---|---|
| Flow cytometer | Lasers: Filter sets: |
Table 1. Example instrument specifications needed run an eBioscience Essential Phenotyping Kit.
T cell immunophenotyping data
A.
Click images to enlarge
B.
Click images to enlarge
C.
Click images to enlarge
Figure 3. Example data obtained from two flow cytometers. T cells were isolated from PBMCs and then immediately frozen. Thawed T cells were then stimulated and stained according to protocol on all 3 eBioscience phenotyping kits. Cells were then assayed on the Invitrogen Attune NxT Flow Cytometer and LSRII Fortessa flow analyzer by 2 separate users. Typically, 50,000 events were collected for gating and data analysis. Data was exported from both instruments as FCS files and analyzed with FlowJo software. Red boxes indicate cell population of interest. (A) eBioscience Essential Human T Cell Phenotyping Kit (B) eBioscience Essential Human Treg Phenotyping Kit (C) eBioscience Essential Human Th1/Th17 Phenotyping Kit.
Instrument specifications required for analyzing eBioscience phenotyping kits
To run panels within these kits, instruments are required to have a 2–3 laser system with flexible filter configurations. Below is an example instrument configuration achievable for most traditional flow cytometers.
| Instrument | Specification |
|---|---|
| Flow cytometer | Lasers: Filter sets: |
Table 1. Example instrument specifications needed run an eBioscience Essential Phenotyping Kit.
T cell immunophenotyping data
A.
Click images to enlarge
B.
Click images to enlarge
C.
Click images to enlarge
Figure 3. Example data obtained from two flow cytometers. T cells were isolated from PBMCs and then immediately frozen. Thawed T cells were then stimulated and stained according to protocol on all 3 eBioscience phenotyping kits. Cells were then assayed on the Invitrogen Attune NxT Flow Cytometer and LSRII Fortessa flow analyzer by 2 separate users. Typically, 50,000 events were collected for gating and data analysis. Data was exported from both instruments as FCS files and analyzed with FlowJo software. Red boxes indicate cell population of interest. (A) eBioscience Essential Human T Cell Phenotyping Kit (B) eBioscience Essential Human Treg Phenotyping Kit (C) eBioscience Essential Human Th1/Th17 Phenotyping Kit.
The Applied Biosystems PureQuant Assay
- Facilitates regulatory filings—shorten development timelines; established performance specifications following ICHQ2(R1) guidelines
- Proven technology—used for immune cell identification and quantification for over a decade
- Easily standardized—helps enable consistent results across multiple users, instruments, and sites
- Scheduling flexibility—run fresh cells, frozen cells or gDNA at your convenience
Cell therapy systems
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Cell Therapy Systems (CTS) support
Cy™ is a trademark or registered trademark of GE Healthcare.
Not for resale. Super Bright Polymer Dyes are sold under license from Becton, Dickinson and Company.
Brilliant Ultra Violet™ and Brilliant Violet™ are trademarks or registered trademarks of Becton, Dickinson and Company or its affiliates, and are used under license.
For Research Use Only. Not for use in diagnostic procedures.
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