Labovert fs inverted microscope

A small piece of adipose tissue was fixed in 10% formalin for 30 min, and then it was washed two times in PBS for 10 min to remove the fixative solution. The tissue pieces were then placed on a coverslip and incubated with FITC-phalloidin primary antibody (1:100) in PBS for one hour and then washed two times with PBS to visualize the F-actin cytoskeleton. The tissue was placed on a coverslip and mounted using DAPI mounting medium. The tissue was visualized using a Nikon Inverted Fluorescent Microscope (Nikon Instruments, Amsterdam, The Netherlands), and images were captured with 10 and 20× magnification objectives.
SVF and F-SVF freshly isolated cells were plated at a density of 2000 cells/10 cm² in expansion medium consisting of DMEM high glucose, 10% foetal bovine serum, 2 mM L-glutamine, and 1% penicillin and streptomycin (all reagents from GIBCO, Rodano, Italy) and cultured for 20 days to monitor colony-forming unit (CFU-F) ability. The cells were then fixed in 10% formalin for 10 min, washed in PBS, and colored with methyl violet to visualize colonies. The staining solution was removed, and the fixed colonies were washed two times with PBS. Images of colonies in SVF and F-SVF conditions were captured using a Leica Labovert FS Inverted Microscope (Leica Microsystems, Milano, Italy).

The Senescence-Associated β-Galactosidase assay (SA-β-Gal) was performed on hAECs cultured from P1 to P4. Cells were seeded in 24-well culture plates (5000 cells/cm²) and cultured in basal medium with or without Refeed^®. The SA-β-Gal assay was performed using a commercial kit (Cell Signaling Technology, Danvers, MA, USA) following the manufactured instructions. The staining reaction was stopped after six hours; images of stained hAECs were acquired using Leica Labovert FS inverted Microscope (Leica Microsystems, Wetzlar, Germany) with Leica MC170 HD digital camera (Leica Microsystems, Wetzlar, Germany), and plates were stored at 4 °C in 70% glycerol. The number of blue-stained SA-β-Gal-positive cells in 10 random fields of the wells was counted; data were analyzed using GraphPad Prism 7.04 software. Results are represented as the percentage of blue-stained cells (SA-β-Gal positive cells) ± SD at each passage for both conditions.

To analyze hASC morphology, cells were seeded in a 24-well plate (Corning Incorporated, Corning, NY, USA) at a density of 3500 cells/cm². After 24 h in standard conditions, cells were exposed for 72 h to different final concentrations of OXT (100, 500, and 1000 nM). At the end of the exposure time, cells were observed under a light microscope Leica Labovert FS inverted Microscope (Leica Microsystems, Wetzlar, Germany), and images were acquired with a Leica MC170 HD Imaging System Camera (Leica Microsystems, Wetzlar, Germany).
To evaluate the expression of the OXTR, hASCs were seeded in 25 cm² flasks (Corning Incorporated, Corning, NY, USA) at a density of 3500 cells/cm². After 24 h in standard conditions, cells were treated with OXT 1000 nM for 72 h, and then harvested for RNA isolation. Untreated cells were used as CTR. RNA was retrotranscribed in cDNA and used in qPCR analysis (see Section 4.10).

To investigate the effect of Refeed^® on cell morphology along passages (P0–P4), hAECs were monitored using a Leica Labovert FS inverted Microscope (Leica Microsystems, Wetzlar, Germany). When cells reached confluence, 10 images of the cell monolayer for each condition (Ctrl and Refeed^®) were acquired using a Leica MC170 HD digital camera (Leica Microsystems, Wetzlar, Germany). The acquired images were used to calculate the cell area of hAECs using ImageJ software [38 (link)] (pp. 671–675). For each culture passage, results are reported as the mean of cell area (µm²) ± SD for both hAEC culture conditions.

hWJ-MSCs seeded at density of 3000 cells/cm² in 24-well plates were exposed for 24 h to CB (0.1, 1 and 3 µM) or DMSO. Every experimental condition was assayed in technical duplicate. At the end of the treatment, a morphological analysis was performed, and representative images of cells were acquired under a light microscope (Leica Labovert FS Inverted Microscope; Wetzlar, Germany) with a Leica MC170 HD Imaging System Camera (Wetzlar, Germany).

For cell migration analysis, hWJ-MSCs were seeded into 24-well plates. After cells reached 100% confluence, a scratch, which consists of two straight lines that crossed in the middle of the well, was made by a pipette tip. Then, hWJ-MSCs were cultured for 24 h in absence or presence of CB (0.1, 1, and 3 µM). Images were acquired at 0 and 24 h from the scratch using a light microscope (Leica Labovert FS Inverted Microscope; Wetzlar, Germany) equipped with a Leica MC170 HD Imaging System Camera (Wetzlar, Germany). To evaluate the reversible effect of CB, after 24 h of treatment, the medium was replaced with standard fresh medium in all samples, and then cells were cultured for additional 24 h before images acquisition.

hWJ-MSCs seeded in 6-well plates were exposed for 24 h to CB (range 0.01–5 μM) or DMSO. Each experimental condition was assayed in technical duplicate. At the end of the exposure-time, CTR and treated cells were detached by trypsin-EDTA and counted, as previously described [22 (link)]. Briefly, cells were resuspended in a medium with 50% of Erythrosine B red dye 0.2% (Sigma-Aldrich Co., St. Louis, MO, USA) in PBS. Not stained viable cells and red stained dead cells were manually counted, at least twice for each condition, using the Neubauer hemocytometer (BRAND GmbH, Wertheim, Germany) and a light microscope (Leica Labovert FS Inverted Microscope, Wetzlar, Germany). The total number of viable and dead cells was calculated according to the manufactured instructions.