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.

Keywords: CyTOF; SynTOF; barcoding; mass cytometry; platelet; red blood cell; synaptosome.

Fig. 1:. Pilot barcoding of small, anuclear particles using 2 or 4 complementary barcodes.

For synaptosomes, we increased barcode reagent concentration (A-C) and varied the ratio of barcoded samples (D-F). We also tested barcoding in platelets and red blood cells (RBCs) (G-I). The full lanthanide-based antibody panel used is found in Table S1; modifications are noted below. (A) Four pairs of aliquots from a single synaptosome sample, each containing ~10^6 particles, were mixed with varying amounts of barcoding reagent (1X, 3X, 6X, or 10X) to a final volume of 1 ml. Barcode 1 (B1) samples at each concentration were labeled with the full antibody panel, and B20 samples were labeled with the same panel minus anti-SNAP25. The paired tubes at each barcode concentration were pooled, processed, and analyzed by mass cytometry (CYTOF). (B) Left: Biaxial plots show Pd102 (B1) vs Pd110 (B20) events for each barcode reagent concentration prior to debarcoding (“Barcoded”). Center: Debarcoding to three .fcs files (B1, B20), and “Unassigned” [i.e., showing conflicting barcode patterns]) demonstrated the expected presence (B1) or absence (B20) of SNAP25 signal, plotted here as event count vs. expression of 155Gd_SNAP25. Right: Pd102 vs Pd110 events following concatenation of the B1 and B20 files. (C) Top: From a total of 200,000 events for each concentration tested, the number of assigned events (B1+B20) increased up to 6X barcode concentration, spread approximately equally between B1 and B20. Bottom: The increased event count in B1 (expressed as fold-increase from 1X barcoding reagent) persisted across synaptosome sequential gating steps (described in Fig S1). (D) Four aliquots from a single synaptosome sample, each containing ~10^6 particles, were barcoded with 6X concentration of B7, B8, B9, or B10. Equal (top) or varying (bottom; 1:2:4:8) volumes from each tube were pooled, and the combined samples were labeled with the full antibody panel, processed, and analyzed by CyTOF. Representative plots of signal intensities are shown in Fig. S4. (E–F) Screenshot of output graph (E) and corresponding values (F) of event count vs. sample barcoded with B7–B10 (shown on the x-axis as c7–c10) from equal (top) and varying (bottom) samples. (G) Increased barcode reagent concentration was tested for platelets and RBCs using the same steps as shown in Panel A for synaptosomes (see Figs S2 and S3 for additional details). In brief, three pairs of aliquots from a single platelet or RBC sample were barcoded 1X, 3X, or 6X of non-overlapping barcodes (B2 and B19 for platelets; B3 and B18 for RBCs) and labeled with either the full antibody panel or the panel minus one antibody (anti-CD61 for platelets and anti-CD235ab for RBCs). Paired tubes were pooled, processed, and analyzed by CyTOF. (H). Left: Biaxial plots show Pd102 vs Pd110 events at 6X barcode reagent concentration prior to debarcoding (“Barcoded”) for platelets (top) and RBCs (bottom). Center: Debarcoded files confirm the presence (B2 or B3) or absence (B19 or B18) of antibody signal in the appropriate samples, plotted here as event count vs. 146ND_CD61 for platelets and 175Lu_CD235ab for RBCs. Right: Pd102 vs Pd110 events following debarcoded file concatenation. (I) Ratio of post- (right in Panel H) to pre- (left in Panel H) debarcoding events for the 3 barcode reagent concentrations tested in platelets and RBCs. Synaptosome data is shown for comparison. Data plotted are mean ± SEM, n=3.

Fig. 2:. Human synaptosome barcoding/debarcoding using the full set of 20 barcodes.

(A) 20 synaptosome aliquots from a single synaptosome preparation were individually barcoded with the full barcode kit at 6X concentration each. Ten odd or even numbered samples were pooled. The odd-numbered sample was labeled with the full antibody panel (Table S1) and the even numbered sample with the full panel minus SNAP25. Odd and even samples were pooled before CyTOF analysis. (B) Heat map showing median intensity of palladium isotopes for debarcoded samples with the 6-choose-3 pattern shown for reference. White “+” boxes indicate presence and black “−” boxes indicate absence of the corresponding metal tag. (C) Signal for antibodies against highly expressed antigens, SNAP25 and CD56 (see also Fig S5A). Medians for plus and minus SNAP25: 15.0±0.5 and 0.6 ± 0.1, respectively. Median across all samples for CD56: 23.8 ± 1.0. (D) Same steps as in A, except all 20 tubes were pooled. (E) Sequential gating was applied to the pre- (left) and post- (right) debarcoded data. Details of the gating steps can be found in the Methods and Figure S1. Biaxial plots show Pd102 v Pd110 for each gating step for the two datasets (see Fig. S5B for SNAP25+ and CD47 events). (F) Fold-change from ungated events is plotted for total, SNAP25+ and CD47+ events across sequential gating of debarcoded samples. (G) Cryopreservation of barcoded, antibody-labeled synaptosome preparations: aliquots of barcoded, antibody-labeled samples were either analyzed by CyTOF (Day 0, n=7) or cryopreserved (see Methods), stored at −80, thawed on the indicated day, run, and analyzed as before. (H) Heat map and (I) line graph of representative debarcoded and gated samples show barcode median intensity over days 0 to 32 (see Fig S5C for SNAP25, CD47, and CD56 data).