Biofuge stratos

The formation of fluorescent AGEs in the reaction mixture after 30 days of incubation was measured according to the method of Wrobel et al. [46 (link)]. Briefly, to 1 mL of the reaction mixture, 250 μL of TCA (100%) was added. The resulting mixture was vortexed for 60 s and centrifuged in a refrigerated centrifuge (Biofuge Stratos, Thermo Scientific) at 14,000 rpm for 4 min. The supernatant was collected in a disposable polystyrene cuvette, and fluorescence intensity was read at an excitation wavelength 355 nm and emission wavelength 460 nm using a spectrofluorometer (Wallac 1420 Victor 3 V, Perkin Elmer). The percentage inhibition of fluorescent AGEs formation was calculated as follows: $$\text{Inhibition of fluorescent AGEs}\left(\%\right)=\left[\left(\text{Fluorescence intensity of control}-\text{Fluorescence intensity of sample}\right)/\text{Fluorescence intensity of control}\right]\times 100$$

One gram of oil sample was dissolved in 5 mL of n-hexane, and 5 mL of methanol was added. The mixture was vigorously vortexed and centrifuged at 7500 rpm (Biofuge Stratos, Thermo Fisher Scientific, Waltham, MA, USA) for 5 min (6,170g, max. radius 9.8 cm). The methanolic layer was separated from the lipid phase, and the extraction with a new portion of methanol was repeated. The total phenolic content in the methanolic extracts was determined using a Folin-Ciocalteu’s reagent [16 ]. Briefly, 0.5 mL of the methanolic extract was diluted in water and then the Folin-Ciocalteu’s reagent (0.5 mL) was added. After 3 min, 1 mL of a sodium carbonate solution (20%) was added, and the absorbance was measured at 760 nm after 60 min with the samples standing in the dark. The total phenolics content in each sample was determined using a standard curve plotted for gallic acid. The results were expressed as µg gallic acid per gram of oil.

Blood samples (3–5 ml) were collected in tubes containing EDTA at 8:00 am after an overnight fasting period, immediately put it into a low temperature centrifuge for centrifugation (Biofuge Stratos, Thermo Fisher Scientific, America, 3000×g for 10 min at 4 °C), and plasma were aspirated and stored at − 80 °C until further processing. Blood was also collected to test liver and kidney function, biochemistry, thyroid function, glucose, and sex hormones, to exclude influencing factors and related diseases. Plasma Nesfatin-1 level was measured using the Human Nesfatin-1 Double Antibody Sandwich ELISA Kit (Boster Biological Technology, Wuhan, China. No. EK1138).

A qualitative analysis was made with the aid of immunofluorescence staining following the experimental protocol previously described [40 (link)]. The tissues were rehydrated with different grades of alcohol, blocked with a blocking buffer solution (1% bovine fetal serum (FBS), and incubated separately with primary antibodies anti-E-cadherin and anti-vimentin, followed by treatment with secondary antibodies marked PE and FITC. 4′, 6-Diamino-2-phenylindole (DAPI) (1 mg/mL; 1 : 10000) was used for nuclear staining. After mounting the slides, images were captured using a Leica BM 4000B fluorescence microscope (Germany). The alteration of EMT was evaluated by targeting E-cadherin, vimentin, and CD133 in PCFE alone and in combination with dacarbazine-treated tumor tissues. The mice melanoma tissues were homogenized and lysed (UP200S) in a western blot lysis buffer and centrifuged at 13500 rpm for 15 minutes (Thermo Scientific Biofuge Stratos). The lysate thus obtained was separated on SDS-PAGE and probed with desired primary and secondary antibodies.

The dried stamen or perianth samples (100 mg/sample) were placed in 5 mL quartz tubes equipped with a vapor condenser, then extracted by ultrasound-assisted extraction in 1 mL 90% (v/v) aqEtOH in the USC1200TH ultrasonic bath (Prolabo, Fontenay-sous-Bois, France) following the optimized extraction conditions: 30 kHz frequency at 45 °C for 45 min [33 (link)]. Then, the extract was centrifuged for 15 min at 5000× g (Heraeus Biofuge Stratos, Thermo Scientific, Illkirch, France). The obtained supernatant was filtered using 0.45-μm nylon syringe membranes (Merck Millipore, Saint-Quentin Fallavier, France). Flavonoid enrichment was then performed through the additional DAX-8 (Merck Millipore, Saint-Quentin Fallavier, France) macroporous resin purification step as described in the previously study [33 (link)].

Successively, 3 mL of LeukoSpin^® medium (pluriSelect Life Science Leipzig, Germany), 3 mL of PBMC Spin^® Medium (pluriSelect Life Science, Leipzig, Germany) and 3 mL of lithium-heparinized whole blood as the final layer were stacked in a 15 mL tube. After centrifugation at 756× g for 20 min without braking, using the BioFuge™ Stratos™ (Thermo Fisher Scientific, Waltham, MA, USA), the granulocyte-rich layer was removed according to the manufacturer’s instructions and diluted with 3 mL DPBS^® (Dulbecco’s phosphate-buffered saline modified without calcium, magnesium and chloride, Thermo Fisher Scientific, Agawam, MA, USA). For further usage, cells were then diluted to a concentration of 18 million cells/mL with RPMI and FCS and centrifuged for 5 min at 272× g.

A 100 mg/sample of dried stamen or perianth of N. lotus was placed into the 5 mL quartz tubes, which were equipped with the vapor condenser, and was then used for ultrasound-assisted extraction in the 1 mL 90% (v/v) aqEtOH in USC1200TH ultrasonic bath (Prolabo, Fontenay-sous-Bois, France) using the optimized extraction conditions (30 kHz frequency at 45 °C for 45 min) [11 (link)]. After that, the extracts were centrifuged at 5000× g for 15 min (Heraeus Biofuge Stratos, Thermo Scientific, Illkirch, France). Then, the supernatant obtained from this step was filtered by using a 0.45 μm of nylon syringe membrane (Merck Millipore, Saint-Quentin Fallavier, France). Then, flavonoid enrichment was performed through the additional DAX-8 (Merck Millipore, Saint-Quentin Fallavier, France) macroporous resin purification process that was previously described by Tungmunnithum et al. [11 (link)].