Immunophenotyping Kits for Flow Cytometry

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

Optimized flow cytometry assays workflow 1

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.
Optimized flow cytometry assays workflow 2

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.
Optimized flow cytometry assays workflow 3

Step 3

  • Use instrument and compensation beads to set up flow cytometer for optimal cell population determinations.

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

Antibody Flow Validated Panels

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.

InstrumentSpecification
Flow cytometer

Lasers:
Violet (405 nm)
Blue (488 nm)
Red (633 nm)

Filter sets:
eFluor 450 Pac Blue: 440/50 nm
eFluor 506: 510/50
PerCP-eFluor 710: 710/50 (695/40 acceptable)
FITC: 530/20
PE: 574/26
APC: 660/20

Table 1. Example instrument specifications needed run an eBioscience Essential Phenotyping Kit.

T cell immunophenotyping data

A.

B.

C.

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

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.

InstrumentSpecification
Flow cytometer

Lasers:
Violet (405 nm)
Blue (488 nm)
Red (633 nm)

Filter sets:
eFluor 450 Pac Blue: 440/50 nm
eFluor 506: 510/50
PerCP-eFluor 710: 710/50 (695/40 acceptable)
FITC: 530/20
PE: 574/26
APC: 660/20

Table 1. Example instrument specifications needed run an eBioscience Essential Phenotyping Kit.

Data

T cell immunophenotyping data

A.

B.

C.

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

Learn more about PureQuant Assays

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Not for resale. Super Bright Polymer Dyes are sold under license from Becton, Dickinson and Company.

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For Research Use Only. Not for use in diagnostic procedures.

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