Cell id 20 plex pd barcoding kit

PBMCs were thawed as previously described ^{29 (link)}, then processed and stained according to the Fluidigm PRD017 V3 protocol, as discussed below. First, cells were washed and incubated with cisplatin (final concentration 5μM) for dead cells exclusion. Next, cells were blocked with 0.5mg/mL Human Fc Block (Biolegend) 10 min at room temperature (RT) and stained 30 min at RT with 27 extracellular antibodies (Supplementary Table 1). Then, cells were fixed (2% PFA, 30 min RT), permeabilized with Barcode Perm Buffer (Fluidigm), and barcoded using the Cell-ID™ 20-Plex Pd Barcoding Kit (Fluidigm). Afterwards, they were incubated with Human Fc Block and stained 30 min at RT with 4 intracellular antibodies (Supplemental Table 1) and labeled overnight with 125nM iridium intercalator (Fluidigm). Finally, cells were diluted in EQTM Four Element Calibration Beads (Fluidigm) before acquisition on a Helios^® instrument (Fluidigm).

Synaptosomes, platelets, or RBCs were fixed as previously described by us for synaptosomes (Gajera et al. 2019 (link)). Events were counted before and after barcoding using Moxiflow Flow MXF001 (Orflo Technologies) and adjusted to 1 million events per sample. The commercially available mass-tag cell barcoding reagent kit (based on Zunder et al, 2015 (link)) contains 20 tubes comprising six Pd-based mass-tags arranged into twenty combinations (“6-choose-3,” Cell-ID^™ 20-Plex Pd Barcoding Kit, Fluidigm# 201060). We used these commercially prepared barcode combinations according to the manufacturer’s recommendation for 1–3 million nucleated cells/ml; we refer to this amount as 1X barcoding reagent concentration, which is approximately 300 nM isothio cyanobenzyl-EDTA(Pd) (Zunder et al. 2015 (link)). After barcoding, samples were washed three times with Maxpar Cell Staining Buffer (Fluidigm# 201068). Antibody labeling exactly followed our previously described method (Gajera et al. 2019 (link)). In particular, SNAP25 was chosen because of its central involvement in the regulation of neurotransmitter release (McMahon & Südhof, 1995 (link)) and its presynaptic abundance. We have previously demonstrated that it can be used to reliably detect synaptic particles in mass cytometry (Gajera et al., 2019 (link))