Zetasizer nano s instrument

MB was measured in a Varian V-3900 2T electromagnet using a standard polarization modulation technique38 . A HeNe laser was used (1.5 mW, 632.8 nm) to probe the dispersion contained inside a 5 mm thick optical cell (Hellma) within a temperature-controlled environment. (Cryo-)SEM was performed on a JEOL 6,330 Cryo Field Emission Scanning Electron Microscope at an acceleration voltage of 3 kV in cryo-mode and 10 kV in dry mode. TEM was performed on a JEOL 1,010 Transmission Electron Microscope at an acceleration voltage of 60 kV. For Cryo-TEM a JEOL 2,100 cryo-Transmission Electron Microscope was used. The hydrodynamic radius was determined by Dynamic Light Scattering, performed with a Malvern Zetasizer Nano S instrument.

Hydrodynamic diameters (D_h) and size distributions of the self-assemblies were determined by dynamic light scattering (DLS). The DLS instrumentation consisted of a Malvern Zetasizer NanoS instrument operating at 25 °C with a 4 mW He–Ne 633 nm laser module. Measurements were made at a detection angle of 173° (back scattering), and Malvern Zetasizer 7.03 software was used to analyze the data.
Static light scattering (SLS) measurements were conducted with an ALV CGS3 (λ = 632 nm) at 20 °C. The data were collected from 12° up to 30° with an interval of 2° and from 30° up to 150° with an interval of 10°, calibrated with filtered toluene and filtered water as backgrounds. The refractive index (RI) of the polymer self-assembly in water was measured to be 0.13 mL g^–1.

Plasma isolated from EDTA blood (25 mL) was centrifuged (Thermoscientific Heraeus Megafuge 16R) with 4800× g for 30 min at 4 °C to remove cell debris. Then plasma was diluted 1:1 in ice-cold PBS (Life Technologies) and filtered through a 0.22 µm sterile filter (TPP) and ultracentrifuged (Sorvall discovery M120) at 150,000× g for 8 h. After one washing step in PBS and another ultracentrifugation for 4 h the pellet containing exosomes were resuspended in PBS and protein content was evaluated by the BCA-based protein assay. Exosomes were isolated from cell culture supernatants of 60–70% confluent tumor cell lines, as described above. The amount of Hsp70 derived from exosomes of the FBS in fresh medium was subtracted. Size and uniformity of the exosomes were characterized by dynamic light scattering on a Zetasizer NanoS instrument (Malvern Instruments, Malvern, UK) and by their protein content was determined by Western blot analysis using antibodies directed against β-actin (A228; Sigma-Aldrich), Hsp70 and Grp75 (SPS-825; StressMarq Biosciences Inc.).

To assess sample homogeneity, dynamic light scattering (DLS) data were collected on a Zetasizer Nano-S instrument (Malvern Instruments Canada, Montreal, QC, Canada), equipped with a 633 nm (red) He-Ne Laser and a 173° backscatter detector (19 (link)) from the same hTR 1-20 DNA samples that were used for sedimentation velocity experiments (Supplementary Figure S3). The samples were centrifuged at 13000 rpm for 5 min in an Eppendorf™ MiniSpin™ centrifuge and then filtered through a 0.1 μm Millipore Ultrafree^®-MC filter immediately before transfer to the 3 × 3 mm quartz cell (Hellma Canada Ltd., Markham, ON, Canada). The temperature was equilibrated to 20°C for 5 min before starting the measurements. The small size of the G4 required very long measurement times and only sample concentrations above 1.0 mg/ml concentrations provided enough signal. The sample preparations were highly homogenous and contained only trace amounts of aggregates or higher order oligomers.

Samples were analysed undiluted on a Zetasizer Nano-S instrument at 20 °C (Malvern Instruments., Worcs., UK) using a red laser at a wavelength of 633 nm and a Hellma Quartz-Suprasil cuvette Type 105.251.005-QS. The light path and centre were set at 3 × 3 mm and 9.65 mm, respectively. All data was recorded based on intensity and converted to relative percentage by volume using Zetasizer software v.6.20 and cumulants fit analysis. The stability indicating nature of this method was confirmed through forced degradation studies.

All solutions were centrifuged and filtered prior to DLS analysis of SaBADH^BME(−). A Zetasizer Nano S instrument (Malvern Instruments Inc., Westborough, Massachusetts, USA) and Zetasizer software 7.01 were used to perform measurements and determine the Z-average hydrodynamic radius (R_h), polydispersity index (PdI) and molecular weight (MW) using a globular polymer model. Data were acquired (ten acquisitions of 5 s each for each of the SaBADH concentrations) at 298 K using auto-attenuated He–Ne laser power (wavelength 663 nm) and a solvent refractive index of 1.33. SaBADH was tested at 10, 20, 30, 50, 80 and 100 µM in 10 mM Tris–HCl buffer pH 8.3, 0.5 mM TCEP and supplemented with 0.0, 0.5, 1.0, 1.5, 2.0 or 2.5 M NaCl or KCl. The analyzed data are an average of triplicate measurements over all concentrations of NaCl or KCl.