Computational protocol: Single-Particle Discrimination of Retroviruses from Extracellular Vesicles by Nanoscale Flow Cytometry

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Protocol publication

[…] SORP BD LSRFortessa for small particle detection with a PMT for forward scatter detection. Specifications for laser wavelengths and power are as follows: 405 nm– 50 mW, 488 nm–300 mW, 561 nm–50 mW, and 640 nm–40 mW. Acquisition was done with BD FACSDiva version 8.0.1. BD Coherent Connection software was used for laser power adjustments. Samples, unless otherwise indicated, were acquired on the low sample pressure setting (at 5 turns on the fine adjustment knob), which equated to a measured flow rate of 20 μl/min. This instrument is run with a BD FACSFlow Supply System (FFSS) for day to day acquisition of cells, however for small particle detection, a dedicated steel sheath tank with a 0.1 μm inline filter was used along with a separate waste tank. This was done because we found the FFSS contributed to excess fluctuations in instrument background noise. Surfactant-free, ultra-filtered, low particle count sheath fluid was used for acquisition (Clearflow Sheath Fluid – Leinco). CS&T was run using the dedicated tank to obtain appropriate laser delays for use with the tank, since there is a difference in pressure between the FFSS and the steel tank. Instrument cleaning procedure prior to acquisition: 10 mins distilled water, 30 min FACSClean (BD Biosciences), 10 min distilled water, 60 min 10% Decon™ Contrad™ 70 Liquid Detergent (Thermo Fisher Scientific), and 10 min distilled water. [...] Flow cytometry data was displayed in height for all figures. For small particle analysis, height is the preferred parameter over area. Area is the integrated value of an electronic pulse based on the height and width (time of flight). However, since the particles of interest are very small, the width or time of flight measurements become less precise. This leaves height as the intensity of the signal as the most accurate parameter for analysis of submicron-sized particles.In our instrument platform, side-scatter was chosen over forward-scattered light detection for the approximation of particle sizes. As approximated by Mie Scatter Theory, the angle of light scatter from a particle in the 100–200 nm size range is such that more light is captured at the side-scatter angle rather than forward, whereas with a cell-sized particle (10 μm) the opposite is true,. We found that despite having a PMT for FSC detection, resolution in SSC was still superior for our particles of interest. Flow cytometry data was analysed using Flowjo VX (FLOWJO, LLC). GraphPad Prism v6 was used for the generation of graphs (GraphPad Software). […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Databases DeCoN
Application Flow cytometry
Organisms unidentified retrovirus