Ultrasonic water bath

Approximately 20 g Turkish white cheese sample was blended with liquid nitrogen and ground cryogenically using a Waring Lextra 2 speed blender (East Windsor, NJ, USA) to produce a fine cheese powder.
Water-soluble extracts followed the procedure described by Subramanian et al. (18 (link)) and were prepared by mixing 0.1 g of the cheese powder with 0.5 mL of distilled water. The mixture was sonicated by an ultrasonic water bath (Fisher Scientific, Pittsburgh, PA) for 10 s to extract water-soluble components, and then 0.5 mL chloroform was added to remove the complex fats, vortexed for 30 s, and the mixture was centrifuged at 15,700 × g for 3 min at 25°C (Megafuge 8, Thermo Fisher Scientific, Waltham, MA, USA). Supernatants (200 μL) were mixed with 200 μL absolute ethanol to precipitate the complex proteins in the mixture and centrifuged at 15,700 × g for 3 min at 25°C. The supernatant was employed for further spectroscopic analysis.
Methanol and ethanol extracts were prepared by mixing 0.1 g of the cheese powder with either 1.0 mL of 100% methanol or 100% ethanol solutions. The mixtures were sonicated by using an ultrasonic water bath (Fisher Scientific, Pittsburgh, PA) for 10 s to break down the cheese clumps and improve the extraction of components and centrifuged at 15,700 × g for 3 min at 25°C. The supernatant was employed for further spectroscopic analysis.

Human ASCs were plated at 5 × 10³/cm² and differentiated in chemically defined medium for 28 days. Culture medium and lysed cell supernatant were individually collected on 14 and 28 days. Cells were lysed using 1% Triton-X solution (Sigma-Aldrich, St. Louis, MO). Cell culture lysate was collected by scrapping with a cell scraper (Fisher-Scientific) and briefly sonicated for 10 s using an ultrasonic water bath (Fisher-Scientific) on ice. Total secreted human adiponectin and leptin were measured using ELISA (R&D, Minneapolis, MN) per our prior methods2 (link)10 (link)11 (link)16 (link)17 (link). Absorbance was read using a microplate reader (Biorad, Hercules, CA). DNA content was measured using Quant-iT™ PicoGreen^® dsDNA Assay Kit (Life Technologies) per our prior methods. A high-range standard curve of 1 μg/mL was used for measuring the DNA concentration of the samples per our prior methods. The samples were prepared in a 96 well black bottom plate (Fisher-Scientific) with absorbance read using microplate reader and standard fluorescein wavelengths (excitation ~480 nm, emission ~520 nm; Molecular Devices, Sunnyvale, CA). Total triglycerides and glycerol contents were measured by processing the sample lysate with Free Glycerol Determination kit (Sigma-Aldrich) per our prior methods2 (link)10 (link)11 (link)16 (link)17 (link).

Sulfated lactosyl archaeol (SLA; 6′-sulfate-β-D-Galp-(1,4)-β-D-Glcp-(1,1)-archaeol) was synthesized, as described previously [20 ]. Cy5.5-OVA conjugate was purchased from Nanocs (New York, NY, USA), while CellVue^TM NIR815 was purchased from Thermofisher Scientific (Waltham, MA, USA).
Thin-film hydration archaeosomes were prepared, as previously described [10 (link)]. In brief, SLA lipid that was dissolved in chloroform/methanol underwent solvent removal under N₂ gas with mild heating to form a thin lipid film. Dried lipids were then hydrated in Milli-Q water for more than 12 h to form a uniform lipid suspension. Next, sonication was applied at 40 °C in an ultrasonic water bath (Fisher Scientific, Ottawa, ON, Canada) for up to an hour until the desired particle size (~100 nm) was obtained. After that, 10 × PBS (Millipore Sigma Canada, Oakville, Ontario) was added and archaeosomes stored at 2–8 °C until used. Note that we have previously demonstrated that archaeosomes stored under similar conditions were stable up to 6 months of testing, which was well beyond the storage time of ~1 month in the current study [21 (link)]. On the day of immunization, antigen solution (ovalbumin; OVA; type VI, Sigma-Aldrich, St. Louis, MO, USA) was added to the pre-formed archaeosomes to reach a desired final concentration.