Determining Nanoparticle-Induced Toxicity in Neurons Using Microglia as a Surrogate Biosensor

Take microglial cultures in a multi-well plate containing media.

Replace half of the media in the test well with media containing silver nanoparticles, and in the control well with vehicle media. Incubate.

The microglia internalize the nanoparticles, become activated, and release pro-inflammatory cytokines.

Transfer the vehicle media and the conditioned media containing these cytokines to filters fitted onto collection tubes.

Centrifuge to remove the nanoparticles, retaining the cytokines.

Invert the filters into new collection tubes, centrifuge, and collect the media. Add fresh media.

Next, take hypothalamic neuronal cultures.

Replace half of the control well media with vehicle media and the test well media with conditioned media. Incubate.

The cytokines bind to receptors on hypothalamic neurons, activating signaling pathways that induce neuronal death.

Add resazurin and incubate.

In live cells, dehydrogenase enzymes reduce the weakly fluorescent resazurin to fluorescent resorufin.

Measure the fluorescence.

Reduced fluorescence indicates increased hypothalamic neuronal death, suggesting nanoparticle-induced neurotoxicity.

To begin, collect 200 microliters of supernatant medium fro