Magnetic Delivery of Antibody-Coated Beads to Live Lymph Node Tissue Slices

Abstract

Abstract Lymph nodes have a high degree of spatial organization, characterized by dynamic zones of cells secreting changing sets of cytokines. Experimental measurement of the local distribution of secreted cytokines in live tissue would provide new insights into the dynamics of cell-cell communication, and may help inform spatially targeted therapies. As a step towards bead-based immunoassays for cytokine detection in situ, here we report our findings for delivering beads uniformly to live tissue, a significant technical challenge due to a dense tissue matrix and potential internalization by endocytic cells. We utilized live slices of lymph node tissue ex vivo to allow access to the interior of the node while maintaining structural integrity and dynamics. Lymph node tissue viability post slicing was confirmed by flow cytometry (80% viable by PI exclusion). We conjugated capture antibodies to magnetic beads using carboxylic acid-amine linker chemistry; linkage was confirmed by a secondary antibody. To test penetrance into a dense matrix, beads with various diameters were delivered to 2 % agarose gel by magnetic force. Large (4 and 6 μm) beads were excluded, while smaller beads (0.22, 0.56 and 1.0 μm) were delivered successfully. Small antibody-conjugated beads also penetrated into live, 300-μm thick lymph node tissue slices, but penetrated only 40 μm deep and were localized to the sinuses. Covalent conjugation of polyethylene glycol (PEG) alongside antibody allowed beads to penetrate up to 70 μm into the tissue and provided a more uniform distribution. Having delivered beads successfully, future work will implement a complete multi-step immunoassay in the tissue.