Mass‐tag barcoding for multiplexed analysis of human synaptosomes and other anuclear events

Abstract

Mass-tag cell barcoding has increased the throughput, multiplexing, and robustness of multiple cytometry approaches. Previously, we adapted mass cytometry for cells to analyze synaptosome preparations (mass synaptometry or SynTOF), extending mass cytometry to these smaller, anuclear particles. To improve throughput and individual event resolution, we report here the application of palladium-based barcoding in human synaptosomes. Up to 20 individual samples, each with a unique combinatorial barcode, were pooled for labeling with an antibody cocktail. Our synaptosome protocol used six palladium-based barcoding reagents, and in combination with sequential gating increased the identification of presynaptic events approximately fourfold. These same parameters also efficiently resolved two other anuclear particles: human red blood cells and platelets. The addition of palladium-based mass-tag barcoding to our approach improves mass cytometry of synaptic particles.

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“…Over five years, 113 synaptosome preparations were made from postmortem human brain collected from carefully annotated adult research participants, 21 of whom met rigorous inclusion/exclusion criteria for both clinical and pathologic features: Controls (n=6), high level AD neuropathologic change (ADNC) only (n=9), and high level LBD only (n=6) ( Table S1 ); note that the ADNC and LBD groups each contained two resilient individuals, meaning high level pathologic change without clinical diagnosis of dementia and/or Parkinson’s disease ( 17 ). Following our established protocol ( 14 ), samples were prepared, barcoded, stained with a 38-plex SynTOF panel ( Table S2 ), and analyzed to detect approximately 100 million events from human synaptosome preparations. Debarcoding and sequential gating ( 14 ) yielded highly enriched single human pre- (n=14,904,100) and postsynaptic (n=823,280) events from Brodmann area (BA) 9 of prefrontal cortex, caudate nucleus, and hippocampus (synaptosome preparations strongly favor pre- over postsynaptic particles).…”

“…Following our established protocol ( 14 ), samples were prepared, barcoded, stained with a 38-plex SynTOF panel ( Table S2 ), and analyzed to detect approximately 100 million events from human synaptosome preparations. Debarcoding and sequential gating ( 14 ) yielded highly enriched single human pre- (n=14,904,100) and postsynaptic (n=823,280) events from Brodmann area (BA) 9 of prefrontal cortex, caudate nucleus, and hippocampus (synaptosome preparations strongly favor pre- over postsynaptic particles). Using the same method and multiplex panel, we also detected >740,000 single presynaptic events from non-human primate (NHP) frontal cortex and striatum, and >440,000 single presynaptic events from cerebral cortex and hippocampus of 22 month-old wild type C57Bl6 and PS/APP transgenic mice ( 18 ).…”

“…The copyright holder for this preprint this version posted June 15, 2021. ; https://doi.org/10.1101/2021.06.14.448240 doi: bioRxiv preprint human protocol as previously described by us (19). NHP samples were prepared for mass cytometry and data acquired and processed exactly as for human samples (14).…”

“…Following our established protocol (14), samples were prepared, barcoded, stained with a 38plex SynTOF panel (Table S2), and analyzed to detect approximately 100 million events from human synaptosome preparations. Debarcoding and sequential gating (14) yielded highly enriched single human pre-(n=14,904,100) and postsynaptic (n=823,280) events from Brodmann area (BA) 9 of prefrontal cortex, caudate nucleus, and hippocampus (synaptosome preparations strongly favor pre-over postsynaptic particles).…”

“…More recently, array tomography (3 to 5 antibody probes) and conventional flow cytometry (2 to 4 antibody probes) have characterized hundreds of thousands to a million individual human synaptic events from synaptosome preparations ( 10–12 ). SynTOF (Synaptometry by Time of Flight) ( 13, 14 ) quantifies tens of millions of individual synaptic events by mass cytometry, permitting robust machine learning (ML) applications. Here, we performed SynTOF on healthy non-human primates, PS/APP mice, and human synaptosomes from carefully selected individuals with only AD neuropathologic change (ADNC) or only Lewy body disease (LBD) ( 15, 16 ), as well as normal age-matched adults.…”