Immunofluorescence analysis of human eosinophils

A prominent inflammatory cell type in allergic diseases is the eosinophil, a granulated white blood cell that releases pro-inflammatory cytokines. Eosinophil-derived cytokines, including interleukin-9 (IL-9) and interleukin-13 (IL-13), can skew the immune response towards an allergic phenotype. Unfortunately, it is challenging to immunolabel and collect quantifiable images of eosinophils given their innate autofluorescence and ability to nonspecifically bind to antibodies. Hence, it is important to optimize permeabilization, blocking, and imaging conditions for eosinophils. Here, we show enhanced protocols to ensure that measured immunofluorescence represents specific immunolabelling. To test this, eosinophils were purified from human blood, adhered to glass coverslips, stimulated with or without platelet-activating factor (PAF), fixed with paraformaldehyde, and then permeabilized with Triton X-100 or saponin. Cells were then blocked with goat serum or human serum and incubated with antibodies labelling cytokines (IL-9 and IL-13) and secretory organelles (CD63 for crystalloid granules and transferrin receptor [TfnRc] for recycling endosomes). Carefully selected isotype controls were used throughout, and cells were imaged using Deltavision super-resolution microscopy. Intensities of fluorescent probes were quantified using Volocity software. Our findings show that permeabilization with saponin, blockage with human serum, and using concentrations of antibodies up to 10 μg/ml allowed us to detect marked differences in fluorescence intensities between isotypes and test antibodies. With the achievement of sufficient qualitative and quantitative measures of increased test probe intensity compared to respective isotypes, these results indicate that our protocol allows for optimal immunolabelling of eosinophils. Using this protocol, future studies may provide further insights into trafficking mechanisms within this important inflammatory cell type.