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It is unknown if surface bound toll-like-receptor (TLR) agonists activate cells via density or total molecular number. To answer this question, we developed a TLR agonist surface conjugated polystyrene microparticle (MP) system. Using a library of MPs with varying TLR agonist density and number, we simultaneously observed innate immune cell MP uptake and TNFα expression using ImageStream flow cytometry on a cell by cell basis. The data shows that total TLR number and not density drives cellular activation with a threshold of approximately 10 5 –10 6  TLR agonists. We believe that this information will be crucial for the design of particulate vaccine formulations.

Determining Whether Agonist Density or Agonist Number Is More Important for Immune Activation via Micoparticle Based Assay