Dmi1 light microscope

Effect of extracts at 50 µg/mL and the 1.56% solution of dental rinse (“Corsodyl 0.2%”, containing chlorhexidine digluconate 0.2% w/v as active agent) on fibroblasts’ motility was investigated using a cell migration-based wound healing assay. The most characteristic readout of this in vitro wound healing assay is the change of the cell-covered area (gap closure) over the period of time. The Culture-Insert2-Well (Ibidi, Gräfelfing, Germany) was provided with two reservoirs for culturing cells that were separated by a 500 μm thick wall. The cells were seeded in the reservoirs and cultured until they attached and formed a monolayer. Removal of the silicone insert from the surface resulted in two precisely defined cell patches, which were separated by a zone that had exactly the same width as the separation wall. Cell migration was monitored by taking photos at different time points. Images were captured directly after insert removal and after 24, 48, and 72 h of observation using a Leica DMi1 light microscope (Wetzlar, Germany).

To investigate the capacity of CRC cells to grow under non-adherent conditions, cells were seeded at a density of 1.5 × 10⁶ cells in 10 cm plates. After 24 h of recovery, the cells were treated with increasing concentrations of SARB (10, 20, 50 μM) or Combi 35 µM. Incubation continued for another 48 h, then the cells were washed with PBS, trypsinized and 5 × 10⁴ cells were then seeded in ultra-low attachment (ULA) six-well plates (Corning) in 2 ml of CSC medium (PromoCell) and 40 μl Matrigel with growth factors (Corning). Spheroid formation was monitored 7 days after seeding and then the spheroids were transferred to 15 ml falcon tubes, washed with PBS, trypsinzed, and 2.5 × 10⁴ cells were seeded in ULA six-well plates with 2 ml CSC medium and 40 μl Matrigel. Cells were let grow for another 14 days and fresh medium was added every 2–3 days. At day 21, the spheroid growth was documented by light microscopy using a Nikon Eclipse Ti–S microscope or Leica DMI1 light microscope. The images were then analyzed using a macro (given in SI) which was imported to ImageJ (modified from ref. ^{58 (link)}) to determine spheroid number.

Cells were fixed with 4% PFA w/v for 30 min at RT and washed once with DPBS and 60% v/v isopropanol (Sigma-Aldrich), respectively. Cells were then stained with ORO working solution for 30 min at RT and washed once with 60% v/v isopropanol and distilled water. Brightfield images were taken using 5 x, 10 x and 20 x objectives of a Leica DMi1 light microscope.

Unstained histological sections were subjected to immunohistochemical (IHC) staining to detect and analyze expression of proteins associated with bone, collagen, and vasculature structure formation. OPN and fibronectin (FN) expression correlate to early bone formation and cellular attachment, respectively, while the hematopoietic stem cell marker, CD34, represents angiogenic functions. Paraffin-embedded sections for IHC staining were prepared according to a standard protocol (Abcam IHC Protocol). Briefly, samples were deparaffinized in xylenes and rehydrated using decreasing concentrations of ethanol, ending with washing in distilled water. Antigen retrieval was performed utilizing a heated target retrieval agent (DAKO). Samples were exposed to 1% Triton in PBS and subsequent protein blocking solution prior to addition of primary antibodies. Biotinylated secondary antibody solutions targeting primary antibody host species IgG were followed by the addition of streptavidin-horseradish peroxidase (HRP). A Nova Red (Vector) kit was then utilized to stain HRP-labeled surface proteins for analysis.
Imaging of IHC-stained slides was performed with a Leica DMi1 light microscope at 5x magnification. Captured images were combined utilizing a FIJI stitching plugin, designed by Dr. Preibisch, to generate full tissue section images.

The 3D spheroidal assay was performed using 3D spheroidal 96-well microplates (4515; Corning) as per the manufacturer’s instructions and as described previously [30 (link),31 (link)]. Briefly, 2.5 × 10³ NB cells per well were seeded and incubated for two days or until the spheroid size reached ~300 μm. Similar size spheroids were randomized and treated with increasing concentrations of CI-1040 for 15 days with regular drug replenishment and spheroidal size measurement on every third day. Spheroidal images were captured using a DMi1 light microscope (Leica Microsystems, Buffalo Grove, IL, USA), and spheroidal size was measured using the Leica software suite tools (LASX, Leica Microsystems, Buffalo Grove, IL, USA). Finally, the Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells (3002; Biotium Inc., Fremont, CA, USA.) was used to fluorescent label the live and dead cells, and the number of live cells in the spheroids was quantified by CellTiter-Glo 3D Cell Viability Assay (G968; Promega Corp., Madison, WI, USA) solution, according to the manufactures instructions [30 (link),31 (link)].

Representative light microscopy images of the two investigated cell lines MCF-7 and MDA-MB-231 were taken at ~70–80% confluency to compare the cell morphology using a Leica DMi1 light microscope (inverse, 20× objective HI Plan I, Leica Microsystems). Images of three independent cell culture plates per cell line were acquired.
For fluorescence staining, MCF-7 (3 × 10⁴ cells/well) and MDA-MB-231 (2 × 10⁴ cells/well) cells were seeded in ibidi^® µ-slide 8-well ibiTreat cell culture plates (80826, ibidi^®). After 24 h, both cell lines had reached a confluency of ~80% and were fixed with 4% phosphate-buffered formalin for 20 min, permeabilized with 0.1% Triton^TM X-100 (T8787, Sigma-Aldrich, St. Louis, MO, USA) solution in PBS (P5368, Sigma-Aldrich) for 15 min, and blocked with 1% bovine serum albumin (BSA, T844.2, Roth) solution in PBS. Then, F-actin was stained with AlexaFluor^® 488-conjugated phalloidin (1:80 in PBS, #8878, Cell Signaling), and nuclei were counterstained with Hoechst 33342 (B2261, Sigma-Aldrich). Finally, the cells were covered with PBS solution, and fluorescence images were taken using a Nikon Eclipse Ti-S fluorescence microscope (40× Plan Fluor objective, Nikon).