Rpmi 1640 medium

Approximately 40 mL of peripheral blood from individuals of all groups (AD, PT, and CT) were collected in heparinized tubes, diluted in a proportion of 2:1 (v/v) with phosphate-buffered saline (PBS) (pH 7.2), and submitted to a Ficoll gradient solution (Amersham Biosciences, Uppsala, Sweden). After centrifugation at 400× g for 30 min at 20 °C, the buffy coat containing the peripheral blood mononuclear cells (PBMCs) was obtained. Cells were washed with PBS and submitted to centrifugation twice (300× g for 10 min at 20 °C). PBMCs were resuspended with 2 mL of RPMI 1640 medium (Cultilab, Campinas, SP, Brazil) supplemented with 10% fetal bovine serum (FBS) (Cultilab, Campinas, SP, Brazil) and 1% of antibiotics (100 UI/mL penicillin and 100 μg/mL streptomycin- Sigma, St. Louis, MO, USA). Cell viability was assessed using the Trypan blue dye (Sigma, St. Louis, MO, USA) and counted using a hemocytometer. The final concentration was adjusted to 106 cells for subsequent staining.

Placental tissues were obtained from 24 women after elective cesarean section deliveries (36 to 40 weeks of pregnancy). Exclusion criteria included pre-eclampsia, chronic hypertension, infectious disease including toxoplasmosis, chorioamnionitis, chronic renal disease, cardiac disease, connective tissue disease, pre-existing diabetes mellitus and gestational diabetes mellitus. Placental tissues were placed in ice-cold sterile phosphate-buffered saline (PBS), pH 7.2, to remove excess blood then aseptically dissected using a stereomicroscope to remove endometrial tissue and fetal membranes up to 1 h after collection. Floating terminal chorionic villous explants containing five to seven free tips per explant were collected as described previously
[21 (link),22 (link)]. Explants were added to 96-well plates (one per well) and cultured in complete medium containing RPMI 1640 medium (Cultilab, Campinas, SP, Brazil) supplemented with 10% fetal bovine serum (FBS) (Cultilab), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich Co., St. Louis, MO, USA) for 24 h at 37°C and 5% CO₂.

BeWo cells were commercially obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), while HTR8/SVneo cells were a gift from Dr. Estela Bevilacqua (University of São Paulo, São Paulo, SP, Brazil). Both cells were cultured in RPMI 1640 medium (Cultilab, Campinas, SP, Brazil) supplemented with 100 U/mL penicillin (Sigma Chemical Co., St. Louis, MO, USA), 100 μg/mL streptomycin (Sigma) and 10% fetal bovine serum (FBS) (Cultilab) at 37°C and 5% CO₂.

The excretory-secretory material of T. canis larvae (TES) was obtained according to Savigny (1975) (link) and Savigny et al. (1979) (link), with some modifications. The larvae were grown in RPMI 1640 medium (Cultilab, Campinas, Brazil), with medium changes being made during their cultivation period (supplemented with 25 mM HEPES, 1% glucose, 100 μL/mL penicillin, 100 μg/mL streptomycin, 0.4 μg/mL ofloxacin and 50 μg/mL fungizone). The supernatant was collected, and a 200 mM protease inhibitor (phenyl-methyl-sulfonyl-fluoride) was added (5 µl/ml of collected medium) after the supernatants were frozen at -20 °C in sterile tubes. After the supernatant was filtered through a 0.22 µ Millipore membrane and in sequence through a 10 Millipore membrane. Dialysis was performed in distilled water and centrifuged at 4 °C at 12,000 × g for 60 minutes. The antigen underwent the lyophilization process, and for protein quantification, the antigen was dissolved in free water. The antigen concentration was measured using a BCA kit (Pierce, Rockford, IL, USA).

Promastigotes of L. braziliensis, at 2 × 10⁶ cells/mL, were incubated with different concentrations (3.125 to 50.0 μg/mL) of the R. graveolens crude extract (Rg) or its alkaloid-rich fraction (Rg-FAE) for 72 h at 25°C, according to previously described [14 (link)]. Parasite viability was evaluated by MTT assay, and percentages of the inhibition growth were expressed in comparison with untreated control. For the intracellular amastigote assays, peritoneal macrophages, obtained from BALB/c mice, were added in the RPMI 1640 medium (Cultilab, So Paulo, Brazil) supplemented with 10% FBS at 2 × 10⁶ cells/mL. Adherent macrophages were infected with L. braziliensis promastigotes in the stationary growth phase (MOI = 10) and incubated for 4 h in 5% CO₂ at 33°C. After washing, various concentrations (6.25 to 50.0 μg/mL) of the R. graveolens crude extract (Rg) or its alkaloid-rich fraction (Rg-FAE) were added for 72 h, according to previously described [14 (link)]. The slides were stained with Giemsa, and the number of amastigotes was determined using light microscopy. The results were expressed in percentage of inhibition of the number of amastigotes, compared with untreated control. All procedures were performed in agreement with the Ethical Principles in Animal Research and according to protocols approved by the Ethical Committee for Animal Research (CEUA≠012/2015).

Human peripheral blood mononuclear cells from healthy donors were collected after informed patient consent. Separation of mononuclear cells was performed by gradient centrifugation methods using Ficoll Histopaque-1077 (1.077 g/cm³) (Sigma–Aldrich, Germany) follow the manufacturer's instructions at 400 g for 30 min. The use of human samples was approved by the local Ethical Committee of the University Center of the Grande Dourados under protocol number 123/12.
The Jurkat (human acute T lymphocytic leukemia) cell line was cultured in suspension in RPMI 1640 medium (Cultilab, Brazil) supplemented with 10% fetal calf serum (FBS, Cultilab, Brazil), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere at 37°C under 5% CO₂.