Primary microglial cells were isolated using a magnetic bead separation technique described in our previous publications (90, 91). Primary microglial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)–F12, 10% fetal bovine serum (FBS), 1% sodium pyruvate, 1% glutamine, 1% penicillin-streptavidin, and 1% nonessential amino acids. The wild-type and ASC-CFP microglial cell lines were donations from D. T. Golenbock (University of Massachusetts Medical School, Worcester, MA). The ASC KO macrophage cell line was donated by K. Fitzgerald (University of Massachusetts). The wild-type microglial cell line was characterized by Halle et al. (15) and our group (92) and cultured in DMEM, 10% FBS, 1% glutamine, and 1% penicillin-streptavidin. Treatments were performed in 2% FBS-containing medium. For LPS-priming treatments, cells were treated with LPS (100 ng/ml) for 3 hours. Next, the cells were triple-washed with full serum medium to remove any excess LPS and then treated with 100 μM Mn for 6 to 24 hours, unless otherwise stated. For exosome treatment, cells were treated with an equal number of exosomes from welders and age-matched controls for 24 hours, after which qRT-pCR analysis was performed.

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