Crl 1634

The cytotoxicity of the isolated compounds was tested using the WST-8 (4-[3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt) conversion assay. The cytotoxicity was evaluated on two normal cell lines, namely PBMC and human foreskin fibroblast Hs27 (ATCC^® CRL-1634™, Manassas, VA, USA). PBMCs were obtained from buffy coat of healthy donors (as approved by the ethical committee of the local health center, Azienda Provinciale per i Servizi Sanitari, Provincia Autonoma di Trento) within 4 h from sample collection and isolated as previously described [9 (link)]. Cells were seeded in 96-well culture plates at concentration of 5000 cells/well and incubated for 24 h before treatment with H. mexicanum extracts and isolated compounds. To measure the cytotoxic effects 10 μL of Cell Counting Kit-8 (Sigma-Aldrich, St. Louis, MO, USA) (containing the WST-8 solution) were added to each well and incubated for 2h and 4h for fibroblasts and PBMCs respectively, at 37C before reading the absorbance at 450 nm.
The results were analyzed by Student’s t-test. The data are expressed as the percentage of viability and standard error of the mean.

The Hs27 cells (human fibroblast, ATCC^® CRL-1634™) were cultured in accordance with the ATCC product sheet in DMEM (Dulbecco’s Modified Eagle Medium, ATCC^® 30-2002™), supplemented with 10% fetal bovine serum (Fetal Bovine Serum, ATCC^® 30-2020™), 100 I.U./mL of penicillin and 100 μg/mL of streptomycin (Penicillin-Streptomycin Solution, ATCC^® 30-2300™). Cells were trypsinized (Trypsin-EDTA Solution, ATCC^® 30-2101™) twice a week and seeded at a density of 1 × 10⁶ per T25 cell culture flask (Corning^®, Sigma-Aldrich) and incubated in 37 °C and 5% CO₂ to achieve a confluent monolayer. The RH strain of Toxoplasma gondii (RH-GFP ATCC^® 50940™, highly virulent, haplogroup first; expression of green fluorescent protein) was maintained in the tachyzoite stage, according to the ATCC product sheet, in parasite culture medium, which contains DMEM medium with 3% HIFBS (heat-inactivated FBS; 1 h in 56 °C). Infected cells were incubated in 37 °C and 5% CO₂.

Colorectal adenocarcinoma cell line HT29 (ATCC, HTB-38) and human skin fibroblast cell line Hs27 (ATCC, CRL1634) were obtained from ATCC (Manassas, VA, United States). Cells were cultured in vitro in DMEM medium (Gibco™, Thermo Fisher Scientific, Waltham, MA, United States), supplemented with 10% (v/v) fetal bovine serum (FBS) (Euroclone, Italy), 1% penicillin (100 U/ml)/streptomycin (100 μg/ml) (Euroclone, Italy), and 1% L-glutamine (2 mM) (Euroclone, Italy) at 37°C with 5% CO₂. Cell cultures were refreshed every 2–3 days during sub-culturing. Hs27 cells were used between passage numbers 3 and 25.

We used HaCaT cells, HS27 fibroblasts (FBS), and HUVECs as substitutes for the epithelial cells, FBS and blood vessel endothelial cells present in skin tissue54 (link). The human epidermal keratinocytes (HaCaT, ATCC^®, USA) and human skin Fb HS27 cells (CRL-1634, ATCC^®, USA) were cultured in high-glucose DMEM (WELGENE, South Korea) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY) and 1% penicillin/streptomycin solution (Gibco, Grand Island, NY). The HUVECs (PCS-100-010^™, ATCC^®, USA) were cultured in EGM-2 supplemented with ascorbic acid, vascular endothelial growth factor, 20% FBS, recombinant human Fb growth factor, hydrocortisone, insulin-like growth factor-1, a recombinant analog of human insulin-like growth factor-1, gentamicin–amphotericin, and heparin (Lonza, Walkersville, MD, USA). All experiments used cells from passages 3–10.

In cell cultures treated with silica, several tests were performed to find the concentration of fluxed silica. To ensure at least 80% viability, silica was insufflated using the flow given by no air. The established flow is 40 µg/hour. In the case of silica and ozone, silica was insufflated using the flow of the ozonator set at an ozone concentration of 120 ppb.
Hs27 (human skin fibroblasts, ATCC CRL-1634) and A549 (lung alveolar adenocarcinoma epithelial, ATCC CCL-185) cell lines were purchased from the American Type Culture Collection.
The cells were seeded in monolayer in Dulbecco’s modified Eagle medium (DMEM) with 0.1 mg/mL streptomycin and 100 UI/mL penicillin, 10% bovine fetal serum and 2 mM L-glutamine (SIGMA, Milan, Italy) at a controlled atmosphere with 5% CO₂, 90% humidity and at 37 °C. The cells were plated to the density of 2500 cells/well for MTS viability test and 10,000 cells/cm² for the micronuclei and comet tests. The cells were detached with 0.05% trypsin–0.02% EDTA. All materials were purchased from SIGMA-Aldrich, Merk Life Science S.r.L., Milan, Italy.

The T. gondii DX strain was used to cause chronic parasite infection in experimentally vaccinated and control (PBS) C3H/HeOuJ mice. This strain was maintained in vivo in both C3H/HeOuJ and BALB/c mice. A highly virulent RH strain (50174, ATCC, Manassas, VA, USA) maintained in vivo and a Me49 (50611, ATCC, Manassas, VA, USA) strain maintained in vitro on human foreskin fibroblasts (Hs27, CRL-1634, ATCC, Manassas, VA, USA) were used to obtain water soluble, whole-cell tachyzoite lysate antigens (TLA) with the freeze–thaw technique, as described previously^{42 (link),43 (link)}.