Dulbecco s modified eagle media

Neuro 2a (N2a) cells were purchased from the American Type Culture Collection (ATCC) and cultured in media containing an equal amount of Dulbecco’s Modified Eagle Media (Thermo Fisher Scientific, Hampton, NH, USA) and Opti-MEM Reduced Serum Medium (Thermo Fisher Scientific). Media was supplemented with 5% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Life Technologies, Carlsbad, CA, USA). Cell transfections were conducted using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. The vector expressing 4R/0N human tau P301L has been previously described [88 (link)]. Twenty minutes post-transfection, wild-type human K18 tau fibrils were added to the cell media to achieve a 1 μM concentration. Cells were harvested 48 hours post-transfection followed by detergent extraction as previously described [100 (link)]. Briefly, cells were initially harvested in PBS with 1% protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA) followed by sequential homogenization/extraction in different detergents (NP40 and DOC), yielding the DOC-insoluble protein fraction which was resuspended in 1x Laemmli buffer prepared from a 4x Laemmli buffer stock solution. These samples were analyzed by LC-MS/MS as described above and in Online Resource 1.

4T1 cells were procured from the American Type Culture Collection (ATCC) and grown in 75 cm² culture flasks to 80% confluence in Dulbecco’s Modified Eagle Media with high glucose and glutamine (Thermo Fisher). Cells were then dispersed by removing media, washing briefly with Dulbecco’s PBS and treating with trypsin for 15 min. A modified version of the PBQM812A-1 vector (System Biosciences) was used to insert a cumate inducible expression cassette, along with a puromycin resistance selection marker, into the 4T1 genome using the piggyBAC transposon system. This vector was modified to induce expression of a His-tagged human EGFR fused via to a T2A self-cleaving linker to GFP at its C-terminus. This vector was first complexed with lipofectamine (Thermo Fisher) before being added to the 4T1 cells in suspension. These transfected cells were then plated on 75 cm² flasks and selected for with puromycin (puromycin Dihydrochloride, Thermo Fisher) starting at 1 μg/mL for several passages and ending with 2 μg/mL. Transfected cells were additionally induced via 15 μg/mL cumate (Sigma-Aldrich) added to the culture media and then sorted for GFP expression via FACS. A subpopulation consisting of the top 5% most fluorescent cells were used in all future experiments.

Nanoparticles were synthesized using DI water, Potassium hexacyanoferrate (II) trihydrate (MW 422.39; K₄[Fe(CN)₆]·3H₂O), iron (III) chloride hexahydrate (MW 270.3; Fe(Cl)₃·6H₂O), Poly (ethylenimine) (PEI, MW 2,000, Mn 1,800, 50% w/v in H₂O), acetate buffer (pH 5.2), citric acid, acetone, and ethanol obtained from Sigma-Aldrich (St. Louis, MO, USA). Murine CpG oligodeoxynucleotide (CpG) TLR9 ligand (ODN 1585; Class A) was purchased from InVivoGen (San Diego, CA, USA). Fluorescent antibodies against HMGB1 (EPR3507) and calreticulin (FMC75) were purchased from Abcam (Cambridge, UK). Dulbecco’s Modified Eagle Media, non-essential amino acids, antibiotic/antimitotic, and β-Mercaptoethanol were purchased from Thermo Fisher (Waltham, Massachusetts). Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Flowery Branch, GA). CD28 (37.51) and CD3e (145–2C11) antibodies were purchased from eBioscience (San Diego, CA). TexMACS Medium and mouse Pan T Cell Isolation Kit II were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).