Heraeus multifuge x3r centrifuge

The hot water extraction (HWE) was performed according to a previously reported method with some modifications [31 (link)]. Briefly, the snow chrysanthemum powders (1.0 g) were firstly refluxed with 10 mL of 80% (v/v) ethanol at 80 °C for 2 h to remove most of the small molecules. Subsequently, the snow chrysanthemum polysaccharides (SCPs) were extracted twice with 30 mL of deionized water at 90 °C for 2 h. After centrifugation at 4000× g for 15 min (Heraeus Multifuge X3R Centrifuge, Thermo Fisher scientific, Waltham, MA, USA), the supernatants were combined and concentrated to 1/3 of the origin volume by a rotary evaporator under a vacuum at 60 °C. Furthermore, the supernatants were precipitated with three volumes of 95% ethanol (v/v) overnight at 4 °C. The precipitations were washed with 70% ethanol (v/v) for three times and then dissolved in deionized water. Finally, the crude snow chrysanthemum polysaccharides (SCP-W) were freeze dried and stored at −20 °C for further analysis.

The process of isolation was performed
as described by A. Boyum.^{27 (link)} Briefly, peripheral
blood was withdrawn from fasted healthy adult volunteers (University
Hospital Jena, Germany) and centrifuged to obtain leukocyte concentrates.
These were aliquoted and mixed with 2.5% dextran in PBS. After 45
min, the leukocyte-rich supernatant was transferred to a density centrifugation
medium (Histopaque-1077; d = 1.077) and centrifuged
(200 rpm, 10 min, RT, without brake, Heraeus Multifuge X3R Centrifuge,
Thermo Fisher Scientific).
Peripheral blood mononuclear cells
(PBMC) were concentrated on top of the density medium and separated
from the other cells. Isolated PBMC were washed with PBS twice (1200
rpm, 5 min, 4 °C) and resuspended in 5 mL of PBS.
For the
isolation of PMNLs, contaminating erythrocytes of pelleted
PMNLs were removed by hypotonic lysis. Afterward, PMNLs were washed
with PBS twice (1200 rpm, 5 min, 4 °C) and resuspended in 5 mL
of PBS. A cell counting system (Vi-CellTMXR, Beckmann Coulter) was
used to determine the cell numbers and cell viability. For counting,
the cell suspension was diluted (1:50) and trypan blue staining [0.4%
(v/v), sterile filtered] was used to determine cell viability.

The extraction procedure was adapted from that of Mané et al. (2007 (link)), as described by Pinasseau et al. (2016 (link)). Briefly, frozen skins were ground with liquid nitrogen with a Mortar Grinder Pulverisette 2 (Fritsch, Idar-Oberstein, Germany). One hundred milliliters of of powder were weighed and 500 μL of methanol was immediately added. Then 3.5 mL of acetone/H2O 70/30 (v/v) 0.05% trifluoroacetic acid were added. The mixture was crushed with Precellys (Bertin Technologies, Montigny-le-Bretonneux, France) during three cycles (3 × 40 s each). 3.5 mL were centrifuged with a Heraeus Multifuge X3R Centrifuge (ThermoFischer Scientific, Waltham, USA) (21,320 g, 5 min, 4°C). Aliquots (1 mL) of the supernatant were dried with Genevac (SP Scientific, Warminster, PA, USA).

A volume of 50 mL of nanoparticle solution was prepared in triplicate and the pH was adjusted to 5.9 using 5 M NaOH. The solution was centrifuged in a Heraeus Multifuge X3R centrifuge (ThermoFisher Scientific, Waltham, MA, USA) at 4 °C and 18,000 g for 1 h. Cycles of slow freezing at −20 °C and thawing at room temperature were performed in order to destabilize the nanoparticles and cause their aggregation, hence facilitating the precipitation. Nanoparticle pellets were lyophilized and weighed. The yield was calculated as follows:
$Yield=\frac{M_{NPs}}{M_T}\times 100\%$
where M_NPs is the mass of CS-TPP nanoparticles recovered and M_T is the combined total weight of CS and TPP used in the formulation.

GPWW was prepared by adding 50 g of as-received mixed grape waste (seeds, skin, and stems) into 1500 mL of distilled water and boiling it for 30 min. Subsequently, to remove the coarse solid residual, vacuum filtration was accomplished. Then, the derived aqueous extract was centrifuged with a Thermo Scientific Heraeus Multifuge X3R Centrifuge and stored at −19 °C before its use. The analytical characterization of the obtained extract was performed using HPLC-MS/MS analysis. The quantification of 38 bioactive analytes belonging to different classes of polyphenols was carried out using a modified version of the already-known method presented by Mustafa et al. [36 (link)], as detailed below.

EVs were isolated with a differential centrifugation method, following a modification of a previously described EVs isolation protocol³⁴ . Pleural fluids and lavages were centrifuged by Thermo Scientific Heraeus MultifugeX3R Centrifuge (FiberLite rotor F15-8 × −50c) at 300 × g during 10 min, followed by a centrifugation step at 2500 × g during 20 min and a centrifugation step at 10,000 g during 30 min. After, the supernatant was filtered through 0.22 µm filters (Merck Millipore) and the sample obtained was transferred to ultracentrifuge tubes (Beckman Coulter) and filled with PBS. To finish the centrifuged procedure, two consecutive ultracentrifugation steps at 100,000 g were performen on a Thermo Scientific Sorvall WX UltraSeries Centrifuge with an AH-629 rotor during 2 hours each. At the end, the pellet obtained with the EVs was resuspended in 50 µL of PBS. From those, 5 µL were isolated for nanoparticle tracking analysis (NTA) and quantification, and the rest was frozen at −80 °C with 500 µL of Qiazol for RNA extraction, or with 45 µL of RIPA buffer (5 nM EDTA, 150 mM NaCl, 1% Triton, 20 nM Tris pH8 and 1:200 protein inhibitors) for protein extraction.

EVs were obtained from the SNs of UAs by differential centrifugation, following a modification of a previously described EVs isolation protocol by Thery et al. [26 ]. Briefly, SNs were thawed and diluted in PBS to a final volume of 25 mL. A centrifugation step at 10,000g (4 °C) for 30 min was performed on a Thermo Scientific Heraeus MultifugeX3R Centrifuge (FiberLite rotor F15-8x-50c) to remove cell debris, macroparticles and apoptotic bodies. The resulting pellet enriched in MVs was resuspended in 50 µL of PBS and frozen at −80 °C. Then, the supernatant was transferred to ultracentrifuge tubes (Beckman Coulter) and filled with PBS to perform a first ultracentrifugation step at 100,000g (4 °C) for 2 h on a Thermo Scientific Sorvall WX UltraSeries Centrifuge with an AH-629 rotor. The supernatant of this second centrifugation was the soluble fraction and was frozen at −80 °C. This first pellet was resuspended in PBS and again centrifuged at 100,000g (4 °C) for 1 h. The final pellet enriched in EVs (possibly along with MVs and some remaining apoptotic bodies) was resuspended in 50 µL of PBS. Five microliters from MVs and EVs pellets were reserved at −80 °C for particle size distribution and quantification by nanoparticle tracking analysis (NTA) while the rest of the sample was frozen at −80 °C for protein extraction.