Quantifying Microglia Aβ42 Uptake

Amyloid Beta 1-42 peptide (Aβ42) conjugated to HiLyte 488 (Anaspec, Fremont, CA) was initially resuspended and fibrilized as reported in^{16 (link),17 (link)}. Briefly, Aβ42 was resuspend in 10 nM NaOH (10% of final volume) and then the concentration was adjusted to 1 mg/ml using PBS. Resuspended Aβ42 was frozen at − 20 °C until use. Aβ42 was thawed and incubated at 37 °C overnight (12–14 h) to fibrilize prior to the phagocytosis assay. Aβ42 was added to culture with either young or aged enriched microglia. Duration of culture is noted in each figure. After microglial enrichment and/or phagocytosis assay, samples were stained for flow cytometry using a fixable LiveDead viability stain (ThermoFisher Scientific) and surface stained with the following antibodies: anti-CD45 (clone: 104), -CD11b (clone: M1/70) (Biolegend), and -TREM2 (clone: 237920) (R&D Systems). Aβ42 uptake was evidenced by the presence of HiLyte 488 in microglia (CD45 intermediate and CD11b⁺ cells) and was quantified via flow cytometry. Data was acquired on a FACSCanto flow cytometer from BD Biosciences and analyzed using FlowJo software (Ashland, OR) or an imaging flow cytometer (Amnis ImageStreamX Mark II, Luminex).