Supplementary MaterialsExtended Data Body 1-1: Comparison of FSC signal from neuronal nuclei and 0. heterogeneous populations of submicron particles such as synaptosomes, but many technical challenges arise in these experiments. To date, most flow cytometry studies of synaptosomes have relied on particle detection using forward scatter (FSC) measurements and size estimation with polystyrene (PS) bead standards. However, these practices have serious limitations, and special care must be taken to overcome the poor sensitivity of conventional flow cytometers in the analysis of submicron particles. Technical artifacts can confound these experiments, especially the detection of multiple particles as a single event. Here, we compared analysis of P2 crude synaptosomal preparations from murine forebrain on multiple flow cytometers using both FSC-triggered and fluorescence-triggered recognition. We applied multicolor fluorescent dye-based assays to quantify coincident particle aggregation and recognition, and we evaluated the fake colocalization of antigens in immunostaining analyses. Our outcomes demonstrate that fluorescence correct and triggering dilution can control for coincident particle recognition, however, not particle aggregation. We verified previous studies displaying that FSC-based size estimation with PS beads underestimates natural particle size, and we determined pervasive aggregation in the FSC range examined generally in most synaptosome movement cytometry research. We discovered that examining P2 examples in sucrose/EDTA/tris (Place) buffer decreases aggregation in comparison to PBS, but will not totally get rid of the existence of aggregates, especially in immunostaining experiments. R406 (Tamatinib) Our study highlights challenges and pitfalls in synaptosome flow cytometry and provides a methodological framework for future studies. (Eppendorf 5424R) for 10 min to remove nuclei and cellular debris, yielding a P1 pellet and an S1 supernatant. A crude nuclei preparation was prepared for flow cytometry according to established procedures (Krishnaswami et al., 2016). The P1 pellet was resuspended in ice cold buffer made up of 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris (pH 7.4), 1 M DTT, 0.1% Triton X-100, and 1 M Hoechst 33342 and homogenized again with 10 strokes of the tight pestle to facilitate release of nuclei. The homogenate was rotated for 15 min at 4C, filtered through a 40-m cell strainer cap, and centrifuged at 500 (Eppendorf 5424R) for 5 min to yield a crude nuclear pellet. The S1 supernatant was further centrifuged at 10,000 (Eppendorf 5424R) for 20 min to obtain the crude synaptosome pellet (P2). P2 pellets were cryopreserved by resuspension in R406 (Tamatinib) 4 mM HEPES/0.32 M sucrose buffer + 5% DMSO and slowly frozen to C80C using an isopropanol freezing container. Frozen synaptosomes were used within two months. This protocol, when combined with rapid thawing at 37C on the day of the experiment, has been shown to preserve synaptosome function and morphology (Gleitz et al., 1993; Daniel et al., 2012). After thawing, all experiments were conducted with either PBS (137 mM NaCl, 2.7 mM KCl, 8 mM R406 (Tamatinib) Na2HPO4, and 2 mM KH2PO4) or sucrose/EDTA/tris (SET) buffer (320 mM sucrose, 5 mM Tris, and 1 mM EDTA). Flow cytometry instrumentation and setup All flow cytometry data acquisition was conducted using the instrument software FACSDiva (BD Biosciences). All flow cytometry data analysis including gating, quantification, and generation of density plots/histograms was performed using FCS Express 6 (De Novo Software). Number of events, % of all events, and R406 (Tamatinib) channel statistics (median, geometric mean, SD, FLN R406 (Tamatinib) etc.) for all those gates were exported using Batch Export for further statistical analysis. All data acquisition on LSRFortessa (hereafter Fortessa, BD Biosciences) was conducted using the lowest possible sample pressure settings. Optical configuration employed and fluorophores detected in these channels are summarized.