Phosphate buffered saline pbs

After exposure of astrocytes in 96‐well plates to hyperoxia or normoxia, measurements of reduction in 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT; Sigma‐Aldrich) were performed to estimate cell viability. MTT is reduced by active mitochondria in vital cells and consequently, the amount of formazan is proportional to the number of living cells. MTT was prepared as a 5 mg/mL stock solution in phosphate‐buffered saline (PBS; Biochrom). A quantity of 50 μL of each well was collected and transferred to a corresponding microtiter plate for LDH release assay.
A quantity of 5 μL of MTT was added to each well (final concentration 0.5 mg/mL) for 3 h. The reaction was stopped by 50 μL 10% Sodium Dodecyl Sulfate (SDS; Sigma‐Aldrich). Absorbance was measured after incubation in a microplate reader (Bio‐Rad, Munich, Germany) at 570 nm, with a reference wavelength of 630 nm.

To verify internalization of unlabeled nanoparticles, cells were stained with Prussian Blue and counterstained with nuclear fast red. Briefly, cells were fixed with 4% paraformaldehyde (PFA; AppliChem, Darmstadt, Germany) for 10 min at room temperature (RT) and washed twice with phosphate buffered saline (PBS; Biochrom, Berlin, Germany). Cells were incubated for 20 min in a mixture of 1% HCl (Carl Roth, Karlsruhe, Germany) and 2% potassium ferrocyanide (AppliChem) at equal volumes, washed twice with PBS and counterstained using 1% nuclear fast red (VWR International GmbH) in 5% aqueous aluminium sulfate (AppliChem) solution for 20 min. Cells were washed twice with PBS and once with distilled water. Finally, cells were embedded in mounting medium consisting of 12% poly(vinyl alcohol) (w/v; Sigma-Aldrich Chemie GmbH), 30% glycerol anhydrous (w/v; AppliChem), 0.53 mM phenol (Sigma-Aldrich Chemie GmbH) and 60 mM TRIS (pH 8.5; AppliChem). After polymerization of mounting medium, stained cells were investigated using the microscope Zeiss Axiovert 40 CFL (Carl Zeiss Microscopy GmbH, Jena, Germany).

Actin staining with diamidino-2-phenylindole dihydrochloride (DAPI) counterstain was used to analyze changes in the structure of the cells’ cytoskeleton after particle exposure. For this purpose, the medium was removed, and the cells were washed with phosphate-buffered saline (PBS; biochrom, Berlin, Germany). The following steps were performed protected from light and at room temperature. Before staining, cells were fixed with 4% paraformaldehyde (PFA; pH: 7.0). After 10 min, cells were rinsed with PBS for 30 seconds. Then, the cell membrane was permeabilized by adding a permeabilization buffer containing 0.05% Triton-X (Merck KGaA, Darmstadt, Germany) for 5 min. Osteoblasts were rewashed with PBS for 30 seconds before 100 nM actin staining solution (100 nM Acti-Stain 488 Fluorescent Phalloidin, Cytoskeleton, Denver, CO, USA) was added to the cells for 30 min. After washing three times with PBS, osteoblasts were incubated with DAPI (Merck KGaA, Darmstadt, Germany) for 5 min to counterstain the nuclei of the cells. The staining solution was removed, and cells were washed with PBS and stored at 4°C until microscopic examination.

Biofilms grown on poly-L-lysine-coated glass slides (BD biosciences) were mock treated or treated with 100 μM AI-2 in growth medium in 12-well-plates and carefully washed with 2 x 1 ml phosphate-buffered saline (PBS) (Biochrom) to remove medium and fixed for 1 h with 2% (v/v) glutaraldehyde (Sigma) in PBS at room temperature. Samples were washed twice with PBS to remove excess fixative and subsequently serially dehydrated by consecutive incubations in 1 ml of 25% (v/v) and 50% (v/v) ethanol-PBS, 75% (v/v) and 90% (v/v) ethanol-H₂O, and 100% ethanol (2x), followed by 50% ethanol-hexamethyldisilazane (HMDS) and 100% HMDS (Sigma). The glass slides were removed from the 100% HMDS and air-dried overnight at room temperature. After overnight evaporation of HMDS, samples were placed on specimen mounts (Ted Pella Inc.) and biofilms coated with 80% Pt-20% Pd to 4 nm using a Cressington 208HR sputter coater at 40 mA prior to examination with a Phenom Table-top scanning electron microscope.

All the chemicals and reagents used were of analytical grade. 2, 2–Diphenyl-1-picrylhydrazyl (DPPH), and butylated hydroxytoluene (BHT) were purchased from Fluka, Germany. Acetic acid was purchased from Merck, Germany. 2,2′-Azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, potassium persulfate, dimethyl sulfoxide, lipopolysaccharide from E.coli O55:B5 (LPS), phosphoric acid, N-(1-Naphthyl) ethylenediamine dihydrochloride, sulfanilamide,3-(4,5-Dimethyl-2-thiazolyl)-2,5-dipheyl-2H-tetrazolium bromide or thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich, USA. Dulbecco’s modified eagle medium (DMEM), Foetal bovine serum (FBS), Trypan blue stain 0.4% and Trypsin-EDTA were obtained from Gibco, USA. Hydrochloric acid and isopropanol were obtained from RCI Labscan, Thailand; Phosphate-buffered saline (PBS) was provided by Biochrom, Germany and mouse TNF-α and IL-6 ELISA kits were purchased from ImmunoTools, Germany.

Male and female NOD.CB17-Prkdc^scid/J mice (Charles River, Lecco, Italy) were kept in accordance with guidelines and under approved protocols by the Local Ethical Committee on Animal Experimentation and by the Italian Ministry of Health. Four groups of mice (n = 7/each) were established as follows: (1) sub-cutaneously flank injected (s.c.f.i) with 2 × 10⁶ BxPC-3 (tumor only control group, CTL) in 200 μl Phosphate Buffered Saline (PBS, Biochrom, GmbH, Berlin, Germany); (2) s.c.f.i with 2 × 10⁶ BxPC-3 and, as soon as an appreciable tumor burden appeared (6 days, 0.4–0.5 mm³), treated with multiple (n = 3) peri-tumoral injections of 10⁶ AD-MSC EV (EV group) in 200 μl PBS every 10 days; (3) tumor injected as in (2) but treated with multiple (n = 3) peri-tumoral injections of 10⁶ AD-MSC sTRAIL (sTRAIL group) in 200 μl PBS, (4) tumor injected as in (2) but treated with multiple (n = 3) tail intra-venous (i.v.) injections of rhTRAIL/Apo2 Ligand (Peprotech Inc.): 5 mg/Kg, (125 μg/mouse) in 200 μl PBS (rhTRAIL group). Parameters such as survival and weight were monitored. In all groups, weights were weekly recorded, tumor sizes were measured with a calliper and volumes were calculated as reported^{37 (link)}: volume = length × width²/2. After 38 days, animals were sacrificed and tissues were harvested for histology.