Lymph nodes are secondary organs of the immune system that are highly organized; cells interact with one another by secreting short-range signaling molecules called cytokines. It is important to characterize these chemical signals within the network of the lymph node and not just in bulk solution. Here we investigate a bead-based assay in live lymph node slices that will allow visualization of specific cytokine profiles during immune responses in a spatially relevant way. Initial work focused on the delivery of antibody-conjugated magnetic styrene beads to lymph node slices. In order to determine which size beads would be able to penetrate the tissue, fluorescent beads with diameters of 0.22, 0.56, 1.00, 4.67, and 6.68 μm were delivered first to 4% agarose as a model matrix. Neither 4.67 nor 6.68 μm beads were able to penetrate the agarose matrix and were therefore excluded from further experimentation. To characterize the spread of the beads in tissue, the remaining bead diameters were delivered to stained slices. Antibody-conjugated beads of each size appeared to be internalized by cells, which would preclude detection of extracellular cytokines. Therefore, beads were conjugated with 20-kDa polyethylene glycol (PEG), a standard approach to minimize uptake by cells. When PEGylated beads were delivered to tissue, they appeared not to be internalized by cells, as evident by the small diameter of the regions of fluorescence (~1 µm, similar to the size of each bead) and a uniform dispersal pattern. Beads with a diameter of 0.22 μm were not readily visualized at the magnification required to visualize an entire slice at one time (4 x) and so were excluded from further studies. Future investigations include 2-photon microscopy of the entire tissue to calculate depth of bead penetration, and flow cytometry to confirm that the beads are not endocytosed. <i>In vitro</i> validation of the immunoassay will also be conducted, moving towards an in-tissue immunoassay. This project will provide a new picture of the cytokine profile during immune responses and inform future vaccine and drug design by highlighting more discrete areas of importance to immune responses.

Bead-based cytokine detection in live lymph node slices

254th National Meeting (2017)

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