Single-cell analysis

Single-cell and bulk analyses

 

Multiparametric single-cell flow cytometry analyses enable us to perform a detailed characterization of each cell present in a sample. This has some great implications compared to other methods without the possibility of single-cell resolution; as a simple example could be the stimulation of cells from peripheral blood to measure secretion of the cytokine IFN-γ. This is done on a routine basis using ELISA and the results will be some concentration number of secreted IFN-γ which can be compared to a negative control. The strength of ELISA is the strict quantitative outcome; this method however cannot be used to determine whether common CD4, CD8 T cells or any other cell type capable of producing IFN-γ are responsible for the signal measured upon stimulation. Using flow cytometry we stain directly the IFN-γ in addition to co-staining of the cell subtypes in question, and we are hence able at linking together the frequency of IFN-γ-secreting cells to their phenotype. Further, the single-cell resolution obtained with flow cytometry gives the possibility of differentiating between cells producing high and low amounts of IFN-γ, also a need which cannot be fulfilled using ELISA.

Another comparison could be done with the genomics and transcriptomics. These offer the simultaneous detection of multiple genes and transcriptional profiles, respectively, but still most often not as single cell analyses and always without the possibility of looking into co-expression of the genes detected.

Parallel detection of multiple cell subsets in single-cell resolution

Our flow cytometry platforms offers measuring or sorting up to 18 parameters in parallel, and thus we are on a routine basis making much more complex analyses than the example referred above. Other examples are our assays for detecting antigen-specific T cells along with their maturation phenotype and for the parallel enumeration of multiple myeloid and lymphoid cell subtypes. The different maturation states of T cells can be defined by staining a handful of surface markers, and we take advantage of this in order to define the ratio between different memory subpopulations selectively on a given population of antigen-specific T cells. For the enumeration of myeloid and lymphoid subtypes we have an established assay using 13 parameters. For both of these assays, the individual cell populations can be of minute frequencies and thus will not be readily detected in a bulk format, while this is possible with flow cytometry due to its single-cell resolution.

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ImmuMap - DTU Science Park, Diplomvej 377, 2800 Kgs. Lyngby, Denmark - Tel: +45 6178 1882

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