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Immunophenotyping is the analysis of heterogeneous populations of cells within mouse blood, splenocytes, and thymocytes for the purpose of identifying the presence and proportions of various cell populations. This is usually accomplished by using antibodies to detect specific antigens expressed by the cells. These antigens are usually functional membrane proteins involved in cell communication, adhesion, or metabolism. Flow cytometry is the method of choice for identifying cells within complex populations, as it allows for multiparameter analysis of thousands to millions of cells in a short time. Applications of this technology are used both in basic research and in clinical laboratories.
The Invitrogen™ Attune™ NxT Flow Cytometer is available with up to 4 lasers and 16 detection channels. All configurations show excellent separation of cell populations into subsets for immunophenotyping. There is strong signal separation for more data clarity, and up to 14-color detection can be performed with the automated compensation module. This application note describes the use of the Attune NxT Flow Cytometer for 4-laser and 10-color immunophenotyping analysis of stained mouse splenocytes using a fixation and permeabilization protocol.
C57BL/6 splenocytes were stained with the cell surface antibodies listed above, followed by fixation and permeabilization using the Invitrogen™ Foxp3 Transcription Factor Staining Buffer Kit, a fixation and permeabilization kit designed to work with transcription factors, like Foxp3, and other intracellular markers. Intracellular staining was performed with Foxp3 Anti-Mouse/Rat mAb, PE Conjugate (eBioscience). The following protocol was used for multiparameter analysis on the Attune NxT Flow Cytometer. Please see the user guide for detailed instructions on experiment setup and running samples.
Samples were collected on the Attune NxT Flow Cytometer using the filters shown in Table 1. The gating strategy used in our multiparameter analysis is described in Figure 1.
Figure 1. Multiparameter analysis of murine regulatory T cells and dendritic cells with the Attune NxT Flow Cytometer. (A) Lymphocytes were gated on FSC/SSC parameters, and B220-expressing B cells were omitted from subsequent analysis. Within the B220–, CD45.2+ gate, T cells were analyzed based on their expression of CD3ε. (B) T cells can be separated into two populations based on expression of the co-receptors CD4 or CD8α. Within the CD4+ T cells there is a subpopulation of suppressive regulatory T cells that express the transcription factor Foxp3 and the cell surface marker CD25 (IL-2Rα). (C) Within the CD3– gate there is a rare population of CD11c+, MHCII+ dendritic cells, which are professional antigen-presenting cells that play a role in activating T cells. Splenic dendritic cells can be subdivided further into CD11b+ and CD8α+ dendritic cell subsets. Values noted are percentage of the parent gate (top number, no parentheses) or percentage of the FSC/SSC lymphocyte gate (bottom number, in parentheses).
Dye | Excitation (nm) | Emission (nm) |
---|---|---|
Pacific Blue | 405 | 440/50 |
Pacific Green | 405 | 512/25 |
Pacific Orange | 405 | 603/48 |
FITC | 488 | 530/30 |
PerCP-Cy5.5 | 488 | 695/40 |
PE | 561 | 585/16 |
PE-Cy7 | 561 | 780/60 |
APC | 637 | 670/14 |
Alexa Fluor 700 | 637 | 720/30 |
APC-Cy7 | 637 | 780/60 |
The Attune NxT Flow Cytometer with 4 lasers and 14 colors shows excellent cell population resolution for 10-color mouse splenocyte immunophenotyping experiments. Designing multicolor panels is greatly facilitated with choices of reagents for 4 spatially separated lasers and 14 colors.