Multiple color immunofluorescence for cytokine detection at the single-cell level

Detection of cytokines and identification of the producer cells are essential to define the interplay and the role of distinct leukocyte subsets in the development of immune and inflammatory responses. Several methods used to study cytokine expression are based on detection of the encoding mRNA (Northern blot, RNase protection assay, RT-PCR) or of protein in the supernatant from stimulated cells (ELISA, RIA, ELISPOT). These are simple and useful, but have limitations related to the need of using purified cell populations to precisely define the effector cells, and exception made for RT-PCR and ELISPOT assays, the requirement for relatively large numbers of cells for sufficient resolution. Here we present a method based on immunofluorescence (flow cytofluorimetry) to detect intracellular accumulation of cytokines in mixed leukocyte populations. It has the distinct advantage of: 1. identifying producing cells in mixed cell populations, 2. comparing quantitatively the levels of production within cells in a given population and among different cell subsets, and 3. defining simultaneous production of distinct cytokines by a single cell type. The detailed description/discussion of the method uses natural killer (NK) cells as an example, but this method can be applied to the study of other cell types, and is of special interest/use when analyzing subsets present in very low proportion.