DOI: 10.5281/zenodo.6976794
title: Raw data on dynamic light scattering assessment of small cellular particles isolated from conditioned culture media of Dunaliella tertiolecta and Phaeodactylum tricornutum. Effect of Triton X-100 and temperature

description: Raw data on dynamic light scattering assessment of small cellular particles isolated from conditioned culture media of microalgae <em>Dunaliella tertiolecta</em> (<em>D. tertiolecta</em>)<em> </em>and <em>Phaeodactylum tricurnutum</em> (<em>P. tricornutum</em>)<em> </em>by dynamic light scattering are presented. The samples were prepared as described below: <strong>Cultivation of the algae:</strong> Cultures of <em>D. tertiolecta</em> CCAP 19/22 and <em>P. tricornutum</em> CCAP 1052/1A from the Culture Collection of Algae and Protozoa (CCAP) of SAMS (Oban, Scotland) were grown in artificial seawater (Reef Crystals, Aquarium Systems, France). 22 g of salt was dissolved in one litre of distilled water, sterile filtered (0.2-micron cellulose filters, ref. 11107-47-CAN, Sartorius Stedim Biotech GmbH, Germany), autoclaved, and supplemented with Guillard’s (F/2) Marine Water Enrichment Solution (ref. G0154, Sigma Aldrich, USA). Cultures were grown in a respirometer (Echo, Slovenia) in 0.5-L borosilicate bottles, at 20 °C and 20 % illumination (approximately 250 μmol/m2s) with a 14-hour light / 10-hour dark cycle, with aeration of 0.2 L/min. <strong>Isolation of cellular nanoparticles (NPs):</strong> NPs were isolated by differential centrifugation, using a protocol widely used for the isolation of extracellular vesicles (Théry C, Amigorena S, Raposo G, Clayton A. Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids. Current Protocols in Cell Biology. 2006;30(1). doi:10.1002/0471143030.cb0322s30). Microalgal cells were removed by low-speed centrifugation (300 g, 10 min, 4°C, centrifuge Centric 260R with rotor RA 6/50 (Domel, Slovenia)), using 50 mL conical centrifuge tubes (ref. S.078.02.008.050, Isolab Laborgeräte GmbH, Germany); and 2000 g, 10 min, 4°C (Centric 400R centrifuge with rotor RS4/100 (Domel, Slovenia)), using 15 mL conical centrifuge tubes (ref. S.078.02.001.050, Isolab Laborgeräte GmbH, Germany). Each step was repeated twice. Then, the cell-depleted medium was centrifuged twice at 10 000g and 4°C for 30 min (Beckman L8-70M ultracentrifuge, rotor SW55Ti (Beckman Coulter, USA)), using thin-wall polypropylene centrifuge tubes (ref. 326819, Beckman Coulter, USA) to remove larger cell debris. Finally, cellular nanoparticles were pelleted by centrifugation at 118 000 g and 4°C, for 70 min in the same type of ultracentrifuge and ultracentrifuge tubes. The isolate obtained from about 30 mL of conditioned media was not visible to the eye. For treatment with Triton X-100, the sample was incubated with Triton X-100 at concentration of 0.1%. <strong>Dynamic light scattering: </strong>The average hydrodynamic radius (<em>R</em><sub>h</sub>) of NPs and the average intensity of light (<em>I</em>) scattered from NPs in the samples were assessed for characterization of NPs by Dynamic Light Scattering (DLS). The value of <em>I</em> was interpreted as a measure of NP concentration (in the case of preserved particle size distribution) or as a topological change (in the case of altered particle size distribution)(Paterna A, Rao E, Adamo G, et al. Isolation of Extracellular Vesicles From Microalgae: A Renewable and Scalable Bioprocess. <em>Front Bioeng Biotechnol</em>. 2022;10:836747. doi:10.3389/fbioe.2022.836747; Brown W, ed. <em>Dynamic Light Scattering: The Method and Some Applications</em>. Clarendon Press ; Oxford University Press; 1993). For analysis of the samples we used Instrument 3D-DLS-SLS cross-correlation spectrometer from LS Instruments GmbH (Fribourg, Switzerland) with a 100 mW DPSS laser (Cobolt Flamenco, Cobolt AB, Sweden) having a wavelength λ<sub>0</sub> = 660 nm. Before measurements, samples were equilibrated in a decalin bath at 25 °C for 15 min. The scattered light was measured at an angle <em>θ</em> = 90° for 120 s. The correlation functions and integral time-averaged intensities <em>I</em>(<em>θ</em>)≡ <em>I</em>(<em>q</em>) (where <em>q</em> is the scattering vector, defined as <em>q</em> =(4π<em>n</em><sub>0</sub>/λ<sub>0</sub>)sin(<em>θ</em>/2), with <em>n</em><sub>0</sub> the refractive index of the medium, in our case estimated by the corresponding value for water, i.e. <em>n</em><sub>0</sub> = 1.33 at 25°C), were recorded simultaneously. The <em>R</em><sub>h</sub> values of NPs were obtained from the diffusion coefficients (<em>D</em>) that were assessed from the correlation function of the scattered electric field (<em>g</em><sub>1</sub>(<em>t</em>)). The <em>g</em><sub>1</sub>(<em>t</em>) function was calculated from the measured correlation function of the scattered light intensity <em>G</em><sub>2</sub>(<em>t</em>) by applying Siegert’s relation (Schärtl W. <em>Light Scattering from Polymer Solutions and Nanoparticle Dispersions</em>. Springer; 2007; Shurer CR, Kuo JCH, Roberts LM, et al. Physical Principles of Membrane Shape Regulation by the Glycocalyx. <em>Cell</em>. 2019;177(7):1757-1770.e21. doi:10.1016/j.cell.2019.04.017). To convert <em>D</em> to <em>R</em><sub>h</sub>, the Stokes-Einstein equation was used (<em>R</em><sub>h</sub> = <em>kT</em>6π<em>ηD</em>, where <em>k</em> is the Boltzmann constant, <em>T</em> is the absolute temperature, and <em>η</em> is the viscosity of the medium in which the particles diffuse). It was assumed that particles have a spherical shape. The viscosity of the medium was not known. We approximated the viscosity value to that of of water at 25°C.To test the effect of Triton X-100 on NPs, 0.1% (V/V) of Triton X-100 was added to the sample before the measurement. The change in <em>R</em><sub>h</sub> distribution and the change of scattered light intensity (Δ<em>I = I</em><sub>sample </sub><em>- I</em><sub>sample<em>+</em>0.1%.TX100</sub>) was determined. Thermal stability analysis was performed using the LitesizerTM 500 instrument (Anton Paar GmbH). Samples were heated from 15 °C to 80 °C in 5 °C steps. When the target temperature was reached the samples were equilibrated for another 5 minutes before 10 measurements of 20 s duration were performed. The size distributions were determined from the mean correlation function using the Anton Paar Kalliope Professional; Version 2.16.0. (Anton Paar GmbH), https://www.anton-paar.com/corp-en/products/details/software-for-particle-analysis-kalliopetm/, applying the Contin approach.
description: We acknowledge grants from Slovenian Research Agency: P3-0388, P2-0232, L3-2621 and J3-3066.