Year: 2019
Type: article
Abstract: Flow cytometry and fluorescence-activated sorting are powerful techniques that hold great promise for studying 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 detection. We implemented multicolor fluorescent dye-based assays to quantify coincident particle detection and aggregation, and we assessed the false colocalization of antigens in immunostaining analyses. Our results demonstrate that fluorescence triggering and proper dilution can control for coincident particle detection, but not particle aggregation. We confirmed previous studies showing that FSC-based size estimation with PS beads underestimates biological particle size, and we identified pervasive aggregation in the FSC range analyzed in most synaptosome flow cytometry studies. We found that analyzing P2 samples in sucrose/EDTA/tris (SET) buffer reduces aggregation compared to PBS, but does not completely eliminate the presence of aggregates, especially in immunostaining experiments. Our study highlights challenges and pitfalls in synaptosome flow cytometry and provides a methodological framework for future studies.
Source: eNeuro
Authors: Benjamin D. Hobson, Peter A. Sims
Institution: Columbia University Irving Medical Center
Language: English
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,925
Awards: Gene Expression Programs in Maturing Dopamine Neurons: Temporal Regulation, Subcellular Localization, and Alteration by Amphetamine, G1405919810, BD Biosciences Influx, Medical Scientist Training Program, Single Cell Analysis of the Infiltrative Margins of Glioblastoma and Post Treatment Recurrence, Cancer Center Support Grant
OpenAlex ID: W2945494438