Immune Netw.  2013 Apr;13(2):43-54. 10.4110/in.2013.13.2.43.

Multiparameter Flow Cytometry: Advances in High Resolution Analysis

• O'Donnell
• Ernst DN
• Hingorani

• KMID: 1431925
• DOI: http://doi.org/10.4110/in.2013.13.2.43

Abstract

Over the past 40 years, flow cytometry has emerged as a leading, application-rich technology that supports high-resolution characterization of individual cells which function in complex cellular networks such as the immune system. This brief overview highlights advances in multiparameter flow cytometric technologies and reagent applications for characterization and functional analysis of cells modulating the immune network. These advances significantly support high-throughput and high-content analyses and enable an integrated understanding of the cellular and molecular interactions that underlie complex biological systems.

Keyword

Antibodies; Multiplex immunoassays; Multi-color flow cytometry; Fluorescent dyes; Immunophenotyping; Single cell analysis/methods

MeSH Terms

Antibodies
Flow Cytometry
Fluorescent Dyes
Immune System
Immunophenotyping
Antibodies
Fluorescent Dyes

Figure

• 

  Figure 1 Polychromatic flow cytometry for analysis of T-cell phenotype and effector function. PBMCs were stained with CD57 FITC, CD11a PE, CD28 PE-Cy™5, CD27 PE-Cy™7, CD8 PerCP-Cy™5.5, CCR7 Alexa Fluor® 647, CD45RA APC-H7, CD3 BD Horizon™ V450, and CD4 BD Horizon™ V500. Cells were then acquired on a BD™ LSR II and analyzed for CD3 and for the subsets within the CD8 T-cell population. These subsets were further analyzed based on CD28 and CD27 staining to obtain the CD27+CD28+, CD27+CD28-, and CD27+CD28- fractions as shown in Panel A. Based on CD197 (CCR7) and CD45RA staining, the cells were then identified as Naïve, Antigen experienced-1, Antigen experienced-2, Antigen experienced-3, and Antigen experienced-4 (Panel B) as described in Appay, et al. (2008). Note that the expression of CD57-positive cells increased with the increased antigen experience. To confirm the increase in antigen experience, CD8 subsets from PBMCs were analyzed after stimulation with PMA/Ionomycin in the presence of BD GolgiStop™ protein transport inhibitor for 5 hours. As expected, there was an increase in the intracellular stain for IFN-γ as the cells moved from the naïve phenotype to the more experienced phenotype.

• 

  Figure 2 Detection of heterogeneous signaling responses within CD4 and CD8 T-cell populations. Human whole blood was stimulated with 0, 1, 10, or 100 ng/ml of recombinant human IL-2 for 15 minutes at 37℃ and fixed, permeabilized, and stained using the BD Phosflow™ human T cell activation kit. Data was acquired on a BD FACSVerse™ flow cytometer and analyzed using Cytobank software. Lymphocyte subpopulations were identified based on surface marker expression (Panel A), and Stat5 (pY694) phosphorylation responses were assessed within each subpopulation (Panel B) or within T cells (Panel C). IL-2 induced a dose-dependent increase in Stat5 (pY694) phosphorylation in T cells and a subpopulation of CD3- lymphocytes. Compared to CD8 T cells, a larger subpopulation of CD4 T cells responded at the lowest concentration of IL-2. However, a subpopulation of CD4 T cells remained unresponsive to IL-2 at the highest concentration, whereas the majority of CD8 T cells responded strongly.

• 

  Figure 3 Multiplex flow cytometric immunoassays. Bead-based multiplex immunoassay principles are demonstrated using the BD™ Cytometric Bead Array (CBA) Flex Set system (Panel A). Each set of capture beads is labeled with two different fluorescent dyes and conjugated to a capture antibody specific for a particular analyte. When mixed with test samples or calibrated standards, the capture beads specifically bind and localize standard or test analytes to their surfaces. PE-labeled detection antibodies bind to another site on the analyte. Excitation of capture bead dyes by the red (635 nm) laser allows the identification of each set of capture beads based on its unique fluorescence intensities in the red (660 nm) and near-infrared (680 nm) channels. Beads with different two-color fluorescence positions can be combined to create relatively high content multiplex assays, such as the 30-plex assay shown (Panel B). Blue-laser (488 nm) excitation of the PE-labeled detection antibodies produces signal intensities commensurate with the amount of bound analyte. The flow cytometric data for each capture bead set can be analyzed to generate standard curves and to quantify the levels of specific analytes in test samples. Standard curves generated from a 15-plex Cytokine BD CBA Flex Set analysis are shown (Panel C).

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