Iscove s medium

The popliteal lymph node cells were fused with myeloma cells (P3X63Ag8.653) at a 5:1 ratio with polyethylene glycol 1500 (PEG 1500; Sigma-Aldrich) following standard procedures by the Washington University Hybridoma Center (43 (link)). Supernatants were harvested for screening when the cells were 50% confluent (about 2 weeks after fusion). Hybridomas were screened for reactivity against C. parvum grown in Caco-2 cells plated on 96-well plates (Greiner Bio-One). Monolayers cells were permeabilized with 0.05% saponin (Sigma) and blocked with a solution containing 0.05% saponin, 5% normal goat serum, and 5% FBS. Hybridoma supernatants were added to the wells in addition to a solution containing anti-RH antibody (rabbit polyclonal sera raised against Toxoplasma gondii strain RH; Covance WU 1047) at a 1:1,000 dilution and 0.02% saponin. After a 1-h incubation, cells were washed and then stained with secondary antibodies conjugated to Alexa Fluor dyes (Thermo Fisher) diluted in 0.01% saponin solution. Positive hybridomas were expanded in Iscoves’ medium (Sigma) supplemented with 20% FBS, cryopreserved in culture medium supplemented with 10% dimethyl sulfoxide (DMSO), and stored in liquid nitrogen.

B-cells and PBMC/CBMC were incubated for 2, 6, 12, 18 or 24 hours (2×10⁵ cells/200 μl in 96-well plates) in Iscove's medium (Sigma-Aldrich) (supplemented with l% glutamine, 1% gentamicin, 1% 2-mercaptoethanol, and 10% FBS) and Poly deoxyadenylic-thymidylic acid sodium salt (poly dA:dT) (10 ug/ml; Sigma-Aldrich), recombinant IFN-α (10 ng/ml; PBL Biomedical Laboratories, Piscataway, NJ, USA), recombinant IFN-γ (10 ng/ml; R&D Systems), anti-IgGAM (2.5 ug/ml; Jackson Laboratories, West Grove, US), CD40L and enhancer (1 ug/ml and 1 ug/ml, respectively; Enzo Life Sciences), anti-IgGAM+ CD40L and enhancer or Lipofectamine2000 Transfection Reagent (Invitrogen). Prior to stimulation, poly dA:dT was complexed with Lipofectamine2000 Transfection Reagent (Invitrogen), according to manufacturer’s instructions. Supernatants were collected after 4 and 24 hours of culture, and stored at -20°C until further use.

BM cells were obtained on day 4 from distinct mice groups: CY–mice immunosuppressed with CY 300 mg/kg (Genuxal) (Baxter Hospitalar Ltda, MG, BRA); CY+Tarin—CY-immunosuppressed mice treated concomitantly with 200 μg tarin on day 0; Tarin—mice treated with 200 μg tarin on the same day or Control—mice inoculated with saline. Cells at 2×10⁵ were plated in double layer soft-agar prepared as described by Heyworth and Spooncer [17 ]. The bottom layer was prepared at a 0.4% final agar concentration in Iscove’s medium (Sigma-Aldrich Co) with 20% FBS, plated in 34-mm TPP tissue culture dishes (Sigma-Aldrich Co). The upper layer containing the cells (0.33% final agar concentration) was supplemented either with 20% supernatants of WEHI and MM3 cells or rHu-G-CSF at 60 μg/plate. Each assay was carried out in duplicate and cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2. The colonies (>50 cells) and clusters (<50 cells) were quantified after 7 days of culture under an inverted microscope.
Filgrastine (Blau Farmacêutica S.A., SP, Brazil) was used as source of recombinant human granulocyte colony-stimulating factor (rHu-G-CSF). The cell lines WeHi 3B and MM3 were obtained from the Rio de Janeiro Cell Bank (APABCAM, RJ, Brazil) and their supernatants were also used as a source of IL-3 and GM-CSF, respectively.