To determine whether the selective uptake of QDs by microglia is influenced by surface chemistry, we used QDs with an amine derivatized polyethylene glycol outer coating that reacts directly with amine reactive Perifosine groups, QDs with a carboxyl coating, or QDs with a PEG coating Inhibitors,Modulators,Libraries conjugated to streptavi din. QD655 PEG, QD655 PEG Strep, and QD655 Carboxy were all selectively taken up by microglia in mixed cortical cultures. We next tested whether the size of QDs affected their ability to enter microglia. QDs of different sizes emit at different wavelengths of the visible spectrum, therefore, QDs of different sizes can be distinguished by their color. We compared QDs emitting at 525, 605, 655, or 705 nm, and found that QD655 was the most efficient at entering microglia.

However, despite their different efficiencies, all QDs tested were selectively taken up by microglia in mixed cortical cultures, regardless of their emission Inhibitors,Modulators,Libraries wavelengths. QD uptake does not affect release of cytokines in primary microglia To determine if uptake of QDs per se alters microglial function, we measured the release of inflammatory cyto kines from primary microglia treated with QDs or with medium alone. QD treatment of primary microglia did not alter their release of tumor necrosis factor a, KC, RANTES, MIP 1a, MIP 1b, or IP 10. Treatment Inhibitors,Modulators,Libraries with QDs also did not change the microglial release of cytokines in response to lipopolysaccharide. The uptake of QDs by microglia depends on clathrin mediated endocytosis We next investigated the mechanism underlying the entry of QDs into microglia.

Because the diameter of QDs Inhibitors,Modulators,Libraries ranges from 10 100 nm, we hypothesized that QDs tra verse the cell membrane via endocytosis rather than pha gocytosis. Since pH sensitivity is considered a good indicator of entry by endocytosis, we examined if the uptake of QDs by microglia depends on pH. The specific inhibitor of endosomal proton ATP pumps bafilomycin elevates the pH in endocytic compartments to neu trality. The entry of QDs into microglia was blocked by BAF in a dose dependent manner, suggest ing that this process depends on the acidification of endosomes. To further dissect the molecular mechanisms underly ing QD endocytosis, we investigated if the entry of QDs depends specifically on clathrin mediated endocytosis, a well studied mode of endocytosis that is crucial for many physiological functions, Inhibitors,Modulators,Libraries including the rapid clear ance and down regulation of activated signaling recep tors and the efficient recycling of synaptic vesicle membrane proteins after neurotransmission.

Cells were treated with CPZ, a cationic amphiphilic drug that prevents the recycling of clathrin and thus prevents endocytosis by clathrin dependent mechanisms. Treatment with CPZ at 10 20 uM significantly inhibited the entry of QDs into microglia. selleck bio Clathrin mediated entry can be also be inhibited by CTB, which induces depolymerization of actin filaments.