In this study, LPS (10 ng/mL, Sigma-Aldrich, St. Louis, MO) and poly(I∶C) (1 µg/mL, Sigma-Aldrich) were used as the agonists for TLR4 and TLR3, respectively, as described [15] (link). Typically, hMSCs are grown to 60–70% confluency in growth medium (DMEM-alpha and 16.5% FCS) prior to the start of an experiment. TLR-agonists are added to fresh growth medium and incubated with the cells for 1 hr. Then the cells are washed twice in growth medium without the TLR-agonists and assayed as described for the experiments. Please note that we believe based on our observations that short incubation (<1 hr) and minimal TLR agonist exposure at concentrations noted above (or less) are essential to arrive at these phenotypes—which mimic the gradient of danger signals endogenous MSCs encounter and respond to at a distance from the site of injury.
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