D pbs

Adipose-derived mesenchymal stem cells (ADMSC; ATCC, PCS-500-011) were grown using the Mesenchymal Stem Cell Basal Medium (ATCC, PCS-500-030), and supplemented with Mesenchymal Stem Cell Growth Kit for adipocyte differentiation- Low Serum (ATCC, PCS-500-040). When cells reached 70–80% confluency, they were seeded at a density of 18,000 cells/cm² and differentiated into mature adipocytes with Adipocyte Differentiation Toolkit (ATCC, PCS-500-050) following the manufacturer’s instructions. Briefly, cells were incubated at 37 °C with 5% CO₂ for 48 h before initiating adipocyte differentiation. Then, media was removed and cell monolayers rinsed with room temperature DPBS (ATCC, 302200). Next, 2 mL (for 6-well plates) of pre-warmed (37 °C) adipocyte differentiation/initiation medium was added to each well to initiate adipocyte differentiation. After a 48 h incubation, half the volume of media was removed and the previous steps repeated for another 48 h. Later, the maintenance phase was initiated by carefully removing 2 mL of media from each well (leaving 1 mL), and replacing it with 2 mL of pre-warmed adipocyte differentiation/maintenance medium in each well. The latter step was repeated every 3–4 days for another 11 days until adipocytes reached full maturity (~12–15 days). Conditioned media (CM) was collected at different time points.

The scratch wound-healing assay was adapted in order to analyze the inhibition of MCF-7 cancer cells migration by modifying the protocol of Governa et al. [85 (link)]. Briefly, MCF-7 cells were seeded into twelve-well cell culture plates (5 × 10⁴ cells/well) and allowed to grow as a monolayer in 37 °C and 5% CO₂. After reaching 90% of confluence, a 200-µL pipette tip was used to scratch two straight lines in the middle of the well. Cells were washed with Dulbecco’s Phosphate Buffered Saline (D-PBS, ATCC, VA, USA) and a fresh medium with 5% FBS and treatments was added to each well. Immediately after scratching and after 12 and 24 h, images were obtained in the same regions with the use of an Olympus IX83 inverted microscope [86 (link)]. The experiments were conducted until the untreated scratched cells, served as control, reached approximately 100% of confluence. All experiments were carried out in triplicate and in two independent series, obtaining six repetitions (n = 6). The images were analyzed by ImageJ, an open-source image processing program, and the percentage of MCF-7 cells migration into the wound was calculated as shown below in Equation (1): $$CellMigration[\%]=\left(\frac{A_{t=0}-A_{t=\Delta t}}{A_{t=0}}\right)\times 100\%$$
where A_t=0 is the initial scratch area and A_t=Δt is the scratch area after n hours of the initial scratch, both in µm².

The unstimulated, stimulated, neutralized, and immunomodulated HEK-Blue^TM cell cultures were trypsinized (trypsin-EDTA, Gibco, Gaithersburg, MD, USA) and removed from the 24-well plates in which they were seeded. After 5 min centrifugation at 200× g, the cell pellet was resuspended in D-PBS (ATCC, Virginia, USA) and incubated for 20 min with FITC anti-TLR2-conjugated monoclonal antibodies (5 µL, Immunostep, Salamanca, Spain). TLR2 expression was evaluated using FACSAria iiu flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) with FACSDiva software (BD Biosciences, Franklin Lakes, NJ, USA). To avoid that the background from the antibody may interfere with the study, samples were washed with D-PBS and centrifuged before being analyzed. Unlabeled and thus non-fluorescent cells, as well as HEK-Blue^TM Null1 cells, were used as both negative controls. TLR2 expression was reported as AFU.