Antibiotic antimycotic solution 100

Ethyl 2-cyanoacrylate (ECA) was used as a monomer for the polymerization. Cardiogreen^® (ICG) and Tween 20 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Phosphotungstic acid was purchased from Carlo Erba reagents s.r.l. (Milan, Italy). Antibiotic/antimycotic solution (100×), containing 10,000 units/mL penicillin, 10 mg/mL streptomycin, 25 mg/mL amphotericin B, and dimethyl sulfoxide (DMSO), were purchased from Sigma-Aldrich (Milan, Italy). Dulbecco’s Modified Eagle Medium (DMEM with 4.5 g/L glucose, l-glutamine, and sodium pyruvate) was purchased from Corning (Mediatech Inc. A Corning Subsidiary Manassas, Manassas, VA, USA). Dulbecco’s Phosphate Buffer Solution and inactivated fetal calf serum were acquired from Biowest (Nuaillé, France). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and trypan blue solution was purchased from Sigma-Aldrich (Milan, Italy). Cell line culture Caco-2 was obtained from the European Tissue Culture Collection. Ultrapure bi-distilled water was obtained by a MilliQ R4 system, Millipore (Milan, Italy). All other chemical reagents were of analytical grade.

A Vero-adapted Ba71V strain was obtained from the African Swine Fever European Union Reference Laboratory (Valdeolmos, Madrid, Spain). A Vero cell line was obtained from ATCC (ATCC^® CCL-81TM) and subcultured in a Minimum Essential Medium (Gibco, Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS, Gibco, Billings, MT, USA) and a 1% antibiotic-antimycotic solution (100×) (Sigma-Aldrich, St. Louis, MO, USA). The cultures were grown at 37 °C, in a humidified atmosphere of air containing 5% CO₂.

Primary HTM cells were isolated from human donor eyes without glaucoma (46, 52, and 55 years old) and characterized as described previously [30 (link)], in accordance with the method of Keller et al. [31 (link)]. HTM cells purchased from ScienCell Research Laboratories (San Diego, CA, USA) were also characterized and used. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotic-antimycotic solution (100×) (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C and 5% CO₂.
To examine the effects of TGF-β3 (Wako Pure Chemical Industries, Ltd., Osaka, Japan), confluent HTM cells from passages 3 to 6 were incubated in a serum-free medium for 24 h. The cells were then treated with different concentrations of TGF-β3 for 72 h, with or without adding Smad3 inhibitor (SIS3) at the same time. The medium was replaced with the serum-free medium for 24 h after exposure. In the control group, the medium was changed at the same time points but only the vehicle was added in place of TGF-β3 or SIS3. All experiments were performed at least three times, and consistency was confirmed using biological replicates.

Primary HCFs were obtained from human donor eyes and characterized as described previously^{38 (link)}. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 containing 10% FBS and antibiotic–antimycotic solution (100×) (Sigma-Aldrich, St. Louis, MO, USA) in a CO₂ incubator at 37 °C. In all experiments, only cells from passages 3–6 were used. To arrest the cell cycle, HCFs were grown to confluence and starved for 48 h in DMEM/F-12 without FBS.

Acetaminophen (cat # A7085) and diclofenac sodium salt (cat # PHR1144) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The V-7 preservation solution was supplied by Vitron (Tucson, AZ, USA) [32 (link)]. Waymouth’s MB 752/1 (without L-glutamine, phenol red and sodium bicarbonate) culture medium and fetal bovine serum were purchased from Invitrogen (Chicago, IL, USA); while L-glutamine, Antibiotic/Antimycotic solution (100×) and gentamicin sulfate were obtained from Sigma-Aldrich (St. Louis, MO, USA). Mixed cellulose-ester IMMOBILIN-NC filters (HATF, 0.45 µm surfactant and triton free, autoclavable), used to support the liver slices on top of the rollers, were obtained from Millipore (Bedford, MA, USA). Dithiobis-nitrobenzoic acid, reduced GSH luciferin, luciferase and ATP were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Anti-CD11b (ICRF44), anti-CD36 (TR9), FcγRIII (3G8), FcγRI (10.1), CD206 (15-2), anti-CD169 (7-239), anti-CD163 (GHI/61), and anti-CD14 (MEM18) were purchased from Exbio (Czech Republic). Anti-CD204-APC (anti-CD204-allophycocyanin) (PSL204) was purchased from Invitrogen, CD206-PE-Cy7 (CD206-phycoerythrin-Cy7) (19.2) from eBioscience, anti-CD68 (298807) from R&D Systems, and anti-CD206 (C10) from Santa Cruz Biotechnology. RNA blue was purchased from TopBio (Czech Republic), Fluoresbrite phagobeads from Polysciences (catalog no. 17153), recombinant human M-CSF from Peprotech (catalog no. 300-25), saponin from Sigma (catalog no. 47036), and human CD14 MicroBeads from Miltenyi Biotec. Albumin fraction V was purchased from Carl Roth (catalog no. 8076). Rp-8Br-cAMPS and 6-Bnz-cAMP were purchased from Biolog (Germany), and antibiotic antimycotic solution (100×) was purchased from Sigma.

Walker 256 mammary gland carcinoma cells (Cell Resource Centre for Medical Research at Tohoku University (CRCTU), Sendai, Japan) were cultured in a 20 mL medium containing RPMI 1640 (Sigma-Aldrich, Steinheim am Albuch, Germany), antibiotic antimycotic solution (100×) (Sigma-Aldrich, St. Louis, MI, USA), glutamine (Merck, Rahway, NJ, USA), and fetal bovine serum (FBS, Capricon Scientific, Ebsdorfergrund, Germany). For the preparation of the medium, a standard bottle (500 mL) RPMI and 5 mL antibiotic antimycotic solution (100×), 5 mL glutamine solution, and 50 mL FBS solution were mixed and dispensed to the culture bottles.
When 80% confluence was achieved, the cells were transferred to a new culture flask. This was done twice a week under the above conditions. To generate a sham group, those cells dedicated for inoculation in sham animals were separated and heat deactivated (10 min/95 °C). To ensure that heat deactivation was successful, a sample of heat-deactivated cells was re-cultured and checked for (the absence of) cell proliferation over two days after deactivation. Heat deactivation functioned in every case, eliminating the need to exclude any sham animals due to methodological deficiencies. Cells of passage 15–20, starting from the supplied cells from the cell bank, were used for inoculation.