Raw data on interferometric light microscopy assessment of small cellular particles isolated from blood plasma, washed erythrocytes, spruce needle homogenate, suspension of flagellae of microalgae Tetraselmis chuii, conditioned culture media of microalgae Phaeodactylum tricornutum and liposomes
Creators: name: Veronika, Kralj-Iglič
name: Arrigler Vesna
name: Apolonija, Bedina Zavec
name: Aleš, Iglič
name: Zala, Jan
name: Matic, Kisovec
name: Ksenija, Kogej
name: Gabriella, Pocsfalvi
name: Marjetka, Podobnik
name: Anna, Romolo
Publisher: name: Zenodo
Year: 2022
Subjects: Interferometric light microscopy
Interferometric nanotracking analysis
Nanoparticle tracking analysis
Concentration of extracellular particles
Size of extracellular particles
Dynamic light scattering
Hydrodynamic radius of extracellular particles
Extracellular vesicles
Liposomes
Exosomes
Flagellosomes
Small cellular particles

description: Samples containing small cellular particles (SCPs) isolated from blood plasma (B<sub>P</sub>), washed erythrocytes (B<sub>E</sub>), spruce needle homogenate (S), suspension of flagellae of microalgae <em>Tetraselmis chuii</em> (T), conditioned culture media of microalgae <em>Phaeodactylum tricornutum</em> (P) and liposomes (L) were assessed by interferometric light microscopy (ILM) (Boccara et al., 2016) for their number density and hydrodynamic radius (R<sub>h</sub>) by using equipment Videodrop (Myriade, Paris, France). Each particle that was included in the analysis was tracked by recording a video and processed individually by using the associated software QVIR 2.6.0 (Myriade, Paris, France). <strong>Sample legend and preparation:</strong> <strong>B1/IS Washed erythrocytes.</strong> Blood was centrifuged for 10 min at 300 g and 18 °C (centrifuge Centric 400/R, Domel, Slovenia) to sediment the erythrocytes. The supernatant from above including the buffy coat was removed. Erythrocytes were resuspended into PBS, mixed by turning the epruvette upside-down and centrifuged for 10 min at 300 g and 18 °C (centrifuge Centric 400/R, Domel, Slovenia). The supernatant was removed and replaced by PBS. Erythrocytes were resuspended in PBS, mixed by turning the epruvette upside-down and centrifuged for 30 minutes at 500g and 18 °C (centrifuge Centric 400/R, Domel, Slovenia). <strong>B2/IS Isolate from plasma.</strong> To obtain plasma, blood was centrifuged for 10 min at 300 g and 18 °C (centrifuge Centric 400/R, Domel, Slovenia) to sediment the erythrocytes. Plasma above the buffy coat was collected into a 4 mL sterilized plastic tube and mixed by turning the tube upside down several times. Plasma was aliquoted into 250 \(\mu \)L samples in Eppendorf tubes and centrifuged at 17,570g and room temperature for 10 minutes in the Centric 200R centrifuge with Lilliput rotor (Domel, Železniki, Slovenia). In each aliquot, upper 200 \(\mu \)L of supernatant was replaced by PBS, resuspended and centrifuged again at 17,570 g and room temperature for 10 minutes in the Centric 200R centrifuge with Lilliput rotor (Domel, Železniki, Slovenia). 200 \(\mu \)L of supernatant was removed and the pellet was suspended in 80 \(\mu \)L of PBS. <strong>B3/SN Supernatant from plasma. </strong>To obtain plasma, blood was centrifuged for 10 min at 300 g and 18 °C (centrifuge Centric 400/R, Domel, Slovenia) to sediment the erythrocytes. 250 \(\mu \)L of supernatant from above the buffy coat was collected into Eppendorf tube and centrifuged at 17,570g and room temperature for 10 minutes in the Centric 200R centrifuge with Lilliput rotor (Domel, Železniki, Slovenia). Upper 200 \(\mu \)L of supernatant was collected to form B3/SN sample. <strong>B4/SN Supernatant from centrifugation of PBS suspension of plasma EPs. </strong>Blood was centrifuged for 10 min at 300 g and 18 °C (centrifuge Centric 400/R, Domel, Slovenia) to sediment the erythrocytes. 250 \(\mu \)L of supernatant from above the buffy coat was collected into Eppendorf tube and centrifuged at 17,570g and room temperature for 10 minutes in the Centric 200R centrifuge with Lilliput rotor (Domel, Železniki, Slovenia). Upper 200 \(\mu \)L of supernatant was replaced by PBS, resuspended and centrifuged again at 17,570 g and room temperature for 10 minutes in the Centric 200R centrifuge with Lilliput rotor (Domel, Železniki, Slovenia). Upper 200 \(\mu \)L of supernatant was collected to form B4/SN sample. <strong>B5/IS, B8/IS Isolates from suspension of aged washed erythrocytes. </strong>Blood was centrifuged for 10 min at 300 g and 18 °C (centrifuge Centric 400/R, Domel, Slovenia) to sediment the erythrocytes. The supernatant from above the buffy coat was removed. The erythrocytes were resuspended into PBS by turning the epruvette upside-down and centrifuged for 10 min at 300 g and 18 °C (centrifuge Centric 400/R, Domel, Slovenia). The supernatant was removed and replaced by PBS. Erythrocytes were resuspended in PBS, mixed by turning the epruvette upside-down and centrifuged for 30 minutes at 500g and 18 °C (centrifuge Centric 400/R, Domel, Slovenia). Supernatant was removed, replaced by fresh PBS and mixed by turning the epruvette upside-down. The washed erythrocytes in buffer were stored at 4 °C. On day 6 after blood collection, the erythrocyte suspension was homogenized by gently inverting the tube 5–10 times. Samples were then subjected to sequential centrifugation of supernatants for 10 min at 500 g, 2000 g, and 4000 g, all at 4 °C in the centrifuge Centric 400/R (Domel, Slovenia). After the last step, the supernatant was subjected to centrifugation at 50,000 g, and 4 °C for 70 min, in an ultracentrifuge Beckman L8–70M with rotor SW55Ti (Thermo Fisher Scientific, USA). The pelleted vesicles were resupended in 5 mL of PBS–citrate and centrifuged at 50,000 g and at 4 °C for 70 min. Pellet was resuspended in 1 mL PBS–citrate to obtain the isolate which was stored at 4 °C until analysed. <strong>S1/IS</strong>, <strong>S6/IS, S9/IS, S11/IS, S12/IS, S13/IS Isolates from spruce needle homogenate. </strong>Branches of spruce were cut from the <em>Picea abies</em> tree and used immediately. Branches were immersed into 1.5 L of water at 30 <sup>o</sup>C with 10 mL of sodium hypochlorite (NaClO, 0.1 %) for 1 hour. The branches were rinsed with water. The needles were cut off from the branches. 50.0 g of wet needles were immersed in 300 mL of ultraclean water and stirred for 1 minute in KOIOS 850W Smoothie Bullet Blender (KOIOS, Neweg, USA). The homogenate was filtered through 0.5 mm nylon net cloth to remove larger particles. EPs were isolated by differential ultracentrifugation. The 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 10 000 g 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, EPs were pelleted by ultracentrifugation at 118 000 g and 4°C, for 70 min in the same type of ultracentrifuge and ultracentrifuge tubes. The pellet was resuspended in 50 µL of ultraclean water. <strong>T1/IS Isolate from suspension of flagellae of <em>Tetraselmis chuii </em>microalgae. </strong> Culture of <em>Tetraselmis chuii </em>CCAP 66/21b 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)18. Cultures were grown in a respirometer (Echo, Slovenia) in 0.5-L borosilicate bottles, at 20 °C and 20 % illumination with a 14-hour light / 10-hour dark cycle, with aeration of 0.2 L/min. Microalgae were harvested when a stable logarithmic growth phase was reached (about one months after inoculation). Flagella were removed from cells by a pH shock: in 10 mL of cell culture, 10 µL of HCl (1 M) was added and distributed over the sample by rapid stirring. Removal of the flagella was confirmed under inverted light microscope (Eclipse TE2000-S, Nikon, Tokio, Japan). Then, 10 µL of NaOH (1 M) was added to the culture to re-establish the initial pH in the culture. EPs were isolated from a 10 mL sample by differential ultracentrifugation, The 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 10 000 g 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, EPs were pelleted by ultracentrifugation at 118 000 g and 4°C, for 70 min in the same type of ultracentrifuge and ultracentrifuge tubes. The pellet was resuspended in 50 µL of initial medium (PBS/ultraclean water/marine water). <strong>P3/IS, P4/IS, P11/IS, P12/IS, P16/IS, P21/IS, P26/IS Isolate from conditioned culture media of <em>Phaeodactylum tricornutum </em>microalgae. </strong>Culture of <em>Phaeodactylum 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)18. Cultures were grown in a respirometer (Echo, Slovenia) in 0.5-L borosilicate bottles, at 20 °C and 20 % illumination with a 14-hour light / 10-hour dark cycle, with aeration of 0.2 L/min. . EPs were isolated from a 10 mL sample by differential ultracentrifugation, The 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 10 000 g 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, EPs were pelleted by ultracentrifugation at 118 000 g and 4°C, for 70 min in the same type of ultracentrifuge and ultracentrifuge tubes. The pellet was resuspended in 50 µL of marine water. <strong>P11/SN</strong> <strong>Supernatant from ultracentrifugation of <em>Phaeodactylum tricornutum </em>microalgae. </strong>Culture of <em>Phaeodactylum 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)18. Cultures were grown in a respirometer (Echo, Slovenia) in 0.5-L borosilicate bottles, at 20 °C and 20 % illumination with a 14-hour light / 10-hour dark cycle, with aeration of 0.2 L/min. . EPs were isolated from a 10 mL sample by differential ultracentrifugation, The 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 10 000 g and 4°C for 30 min (Beckman L8-70M ultracentrifuge, rotor SW55Ti (Beckman Coulter, USA)), using 6 mL thin-wall polypropylene centrifuge tubes (ref. 326819, Beckman Coulter, USA) to remove larger cell debris. Finally, EPs were pelleted by ultracentrifugation at 118 000 g and 4°C, for 70 min in the same type of ultracentrifuge and ultracentrifuge tubes. Supernatant was collected to form P11/SN sample. <strong>LA-1000 liposomes diluted 1000x</strong>. The sample was prepared by mixing 25 weight % of soya granules (Fiorentini, Torino, Italy), 50% of ultraclean water and 25% of glycerol. The soyabean lecithin granules were placed into the falcon tubes. Water was added and the suspension was left at room temperature for 1 hour. Glycerol was added and the sample was mixed by pipetting with Pasteur pipette. The sample was diluted 1000x and filtered through 0.4 \(\mu \)mm filters. <strong>LB-1000 liposomes diluted 1000x. </strong>The sample was prepared by mixing 25 weight % of soya granules (Fiorentini, Torino, Italy), 50% of supernatant of isolation of EVs from spruce needle homogenate and 25% of glycerol. The soyabean lecithin granules were placed into the falcon tubes. Spruce supernatant was added and the suspension was left at room temperature for 1 hour. Glycerol was added and the sample was mixed by pipetting with Pasteur pipette. The sample was diluted 1000x and filtered through 0.4 \(\mu \)mm filters. <strong>LC-1000 Liposomes diluted 1000x. </strong>The sample was prepared by mixing equal weight % of soya granules (Fiorentini, Torino, Italy), supernatant of isolation of EVs from spruce needle homogenate and glycerol. Soyabean lecithin granules were placed into the falcon tube and glycerol was added. The sample was mixed by wortexing and supernatant of isolation of EVs from spruce needle homogenate was added. The sample was mixed by repetitive turning the falcon tube upside down. The sample was kept at room temperature for 6 months. The sample was diluted 1000x and filtered through 0.4 \(\mu \)mm filters. <strong>LA-100 liposomes diluted 100x. </strong>The sample was prepared by mixing 25 weight % of soya granules (Fiorentini, Torino, Italy), 50% of ultraclean water and 25% of glycerol. The soyabean lecithin granules were placed into the falcon tubes. Water was added and the suspension was left at room temperature for 1 hour. Glycerol was added and the sample was mixed by pipetting with Pasteur pipette. The sample was diluted 100x and filtered through 0.4 \(\mu \)mm filters. <strong>LB-100 liposomes diluted 100x.</strong> The sample was prepared by mixing 25 weight % of soya granules (Fiorentini, Torino, Italy), 50% of supernatant of isolation of EVs from spruce needle homogenate and 25% of glycerol. The soyabean lecithin granules were placed into the falcon tubes. Spruce supernatant was added and the suspension was left at room temperature for 1 hour. Glycerol was added and the sample was mixed by pipetting with Pasteur pipette. The sample was diluted 100x and filtered through 0.4 \(\mu \)mm filters. <strong>LC-100 Liposomes diluted 100x</strong>. The sample was prepared by mixing equal weight % of soya granules (Fiorentini, Torino, Italy), supernatant of isolation of EVs from spruce needle homogenate and glycerol. Soyabean lecithin granules were placed into the falcon tube and glycerol was added. The sample was mixed by wortexing and supernatant of isolation of EVs from spruce needle homogenate was added. The sample was mixed by repetitive turning the falcon tube upside down. The sample was kept at room temperature for 6 months. The sample was diluted 100x and filtered through 0.4 \(\mu \)mm filters.
description: We are indebted to Slovenian Research Agency for grants P2-0232, P3-0388, P1-0201, J3-3066, L3-2621 and IO-0003.
description: {"references": ["46.\tBoccara, M.; Fedala, Y.; V\u00e9nien-Bryan, C.; Bailly-Bechet, M.; Bowler, C.; Boccara, A.C. Full-field interferometry for counting and differentiating aquatic biotic nanoparticles: from laboratory to Tara Oceans. Biomed. Opt. Express 2016, 7, 3736-3746. https://doi.org/10.1364/BOE.7.003736"]}