Analyzing Single Cell Secretions by "Shadow Imaging"

Here, we describe a method, which we term "shadow imaging," to analyze the secretions of individual cells at immune synapses or other cell contacts. Following immune synapse formation and cellular activation on ligand-rich slides, the position of each cell is recorded using a pulsed immunofluorescence stain against the proteins on the ligand-rich slide surface. The pulsed stain does not penetrate the synaptic cleft, resulting in an unlabeled region or "shadow" beneath cells that is retained following cellular detachment. The secreted components, such as perforin, exosomes, or other types of extracellular vesicles, are retained on the slide and can be analyzed on a single-cell basis using immunofluorescence. The ability to identify single cells secreting different combinations of particles, proteins, and vesicles enables us to better understand the heterogeneity in immune cell secretions and can be used as a novel approach for phenotyping cell populations.