Infinite 200 pro

The wells were fixed by removing the culture media, washing twice with PBS, adding 3.7% formaldehyde for 20 minutes, then washing twice in diH₂O. The calcium quantification was performed by Alizarin Red S (ARS) staining. ARS was dissolved at 1 w/v% in diH2O, filtered (0.45 µm), and the pH was adjusted to 4.1. The wells were stained for 30 minutes before washing away residual ARS with diH₂O. The wells were air-dried and photographed before destaining with 1 mL of 5% perchloric acid. 150 µL was then transferred in triplicate to a 96-well plate and read at an absorbance of 405 nm. (Tecan infinite 200-pro). The concentration of ARS was determined via a standard curve.
After calcium staining, the wells were washed again with diH₂O before staining for collagen with direct red 80 (DR80). DR80 was dissolved in saturated picric acid at 0.1 w/v% and filtered (0.45 µm). The wells were stained for 1 hour before washing away residual DR80 with diH2O. The wells were air-dried and photographed before destaining with 1 mL of 0.2 M sodium hydroxide:methanol in 1:1 ratio. 150 µL was then transferred in triplicate to a 96-well plate and read at an absorbance of 540 nm. (Tecan infinite 200-pro). The concentration of DR80 was determined via a standard curve.

BC cell viability in the presence of OB-derived CM or 0,1% α-MEM was assessed by AlamarBlue® assay according to manufacturer's instructions (Invitrogen, Darmstadt, Germany). Briefly, cells were cultured for three days, then incubated with AlamarBlue for 4 hours and their absorbance measured with a plate reader at 570 nM with wavelength correction at 600 nm (Infinite 200 PRO, Tecan, Männedorf, Swiss).
The cytotoxic effect of INCB28060 on BC cell lines was evaluated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma Aldrich, Schnelldorf, Germany). Cells were seeded in 96-well microplates. After 72-hour incubation, 10 μL of MTT solution was added to each well and the plates incubated for 4 hours at 37°C. The optical density was measured in the linear range using a microplate reader at 570 nm with a wavelength correction at 630 nm (Infinite 200 PRO, Tecan, Männedorf, Swiss).

C-reactive protein (CRP) was measured by nephelometry. The amount of ecDNA in plasma was quantified with the Quant-iT PicoGreen dsDNA Assay-Kit (#P11496; Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Briefly, samples were diluted 1:20 in a TE buffer and Quant-iT PicoGreen dsDNA reagent was added in a 1:1 ratio followed by incubation for 5 min at room temperature in the dark. Fluorescence was measured in an Infinite 200 PRO plate reader (Tecan, Männedorf, Switzerland) with excitation at 485 nm and emission at 535 nm. The concentration of the ecDNA was calculated with the standard provided by the kit. NE activity was assessed in plasma with the fluorogenic substrate MeOSuc-AAPV-AMC (sc-201163; Santa Cruz Biotechnology, Dallas, TX, USA) to a final concentration of 100 μM at 37 °C. Fluorescent readings at 37 °C were collected on a TECAN Infinite 200 PRO using the filter set ex. 360 nm, em. 465 nm for 24 h in a 20 min interval.

HEK293T cells at a density of 10⁴ cells/well were seeded in 96-well plates and incubated at 37 °C for 12 hours, 5% CO₂. The preformed human Tau aggregates at a concentration of 10 μM were added in each well and the basic limonoid compounds (gedunin, azadirone, azadiradione and epoxyazadiradione) at 1, 2, 5, 10, 20, 50 μM concentrations were added with only human Tau aggregates as a negative control at final volume of 100 μL and incubated for 24 hours at 37 °C, 5% CO₂. After 24 hours, MTT reagent was added at a concentration of 0.5 mg/mL per well and incubated for 3 hours at 37 °C, 5% CO_2. Adding DMSO solubilized the formazan and the absorption at 570 nm measured in spectrophotometer (Tecan Infinite 200 PRO multimode micro plate reader). Limonoids effectively prevent Tau aggregation and thus led to the formation of distinct Tau species as observed by TEM. The toxicity of the resultant Tau species were studied by MTT assay, where 10,000 cells were seeded per well. After 24 hours, cells were treated with the reaction mixture at 5 μM Tau concentration. MTT was added at a concentration of 500 μg/mL post-incubation for 24 hours. After addition of MTT the cells were further incubated for 3 hours. The resultant formazan is dissolved by adding 100 μL DMSO. The obtained purple colour complex is read at 570 nm in spectrophotometer (Tecan Infinite 200 PRO multimode micro plate reader).

RAW 264.7 cells were
seeded in a 96-well plate at a density of 1 × 10⁴ cells
per well. Then, the cells were treated with BSA (9 μg/mL), R-BSA
(11 μg/mL), PSiNPs (100 μg/mL), BSA@PSiNPs (100 μg/mL),
R-BSA@PSiNPs (100 μg/mL), LPS (100 ng/mL) and IFN-γ (25
ng/mL), or IL-4 (25 ng/mL) for 24 h. Because NO molecules are very
unstable in aqueous solution to form nitrite, cell culture supernatants
were collected and quantified spectrophotometrically using the Griess
reagent (Beyotime Biotech, China) to analyze the NO amount. The absorbance
intensity of 540 nm was measured by multifunctional enzyme marker
(Infinite 200Pro, Tecan, Swiss). The relative NO production was calculated
according to the following formula:
To confirm the production of the TNF-α,
the cell culture supernatants were assayed using precoated enzyme
linked immunosorbent assay (ELISA) kits (MultiSciences Biotech, China)
following the manufacturer’s instructions. The absorbance intensity
at 450 and 630 nm was measured by multifunctional enzyme marker (Infinite
200Pro, Tecan, Swiss). And the final absorbance intensity = absorbance
intensity at 450 nm – absorbance intensity at 630 nm. Finally,
the relative TNF-α production was calculated according to the
following formula: