Opteia set

Supernatants were collected from cells at the indicated time points. IL-10 and IL-6 were quantified by ELISA (human IL-10 and IL-6 ELISA OptEIA Sets, BD Bioscience, Heidelberg, Germany). Human immunoglobulins were quantified using Human IgG/IgM/IgA ELISA Quantitation Sets (all from Bethyl Laboratories, TX, USA).

Individual mouse spleen was collected 1 month after challenge from the immunized and naïve groups. Single-cell suspensions were prepared from each spleen. Cells were incubated in 96-well flat culture plates for 3–4 days at 37°C in the presence of 5% CO₂. Cells in 100 μl of RPMI-1640 were stimulated with 100 μl of 0 or 2 μg/ml T. gondii RH. For the cytokine assay, supernatants of spleen cell cultures were collected from each well by separation and stored at -20°C until use. OptEIA sets (BD Bioscience, San Jose, CA, USA) were used to determine the concentration of interferon-gamma (IFN-γ), interleukin (IL)-6 and IL-10 in culture supernatants following the manufacturer’s procedures. To determine CD4+ and CD8+ T cells, single-cell suspension from spleen were stained with CD8 (BD Biosciences) and analyzed using a FACScan flow cytometer (BD, Mountain View, CA, USA). Results were analyzed using WinMDI 2.9 software (De Novo Software, Los Angeles, CA, USA).

Cytokine levels in cell culture supernatants were measured by sandwich ELISA. Microtiter plates (Costar EIA/RIA plates, Corning Inc.) were coated with a cytokine-specific antibody. Expression levels of IL-6, IL-10, and IL-12p40 were measured according to the manufacturer’s instructions (OptEIA Sets, BD Biosciences). TNF-α level was measured according to the manufacturer’s instructions (ELISA Ready-Set-Go, eBioscience). In all cases, 10% FCS in PBS was used as a blocking solution. TMB substrate solution (BioLegend) was used to develop a colorimetric reaction, which was stopped with 2 M sulfuric acid. Optical density was measured at 450 (570) nm using a microtiter plate reader (PowerWaveX, Bio-Tek Instruments).

BLS was administered 2 or 10 days after tumor inoculation. Treatment was also performed in healthy mice. Level of IFN-γ was measured in serum 3 h after BLS injection, using ELISA (all OptEIA sets; BD Pharmingen), following the manufacturer’s instructions. The reaction was developed by adding 50 µl of a solution containing 2 g/l ortho-phenylenediamine and 0.03% H₂O₂ in 0.1 M citrate–phosphate buffer and was stopped with 50 µl of 4 N H₂SO₄. The final color was read at 492 nm in an ELISA reader (SLT Lab Instruments). The detection limit was 31.3 pg/ml.

DC2.4 cells and RAW264.7 cells were seeded on 96-well plates at a density of 5 × 10⁴ cells/well and incubated for 24 h before treatment. Then, ORN-1, hexapodRD6, or RDgel diluted in 0.1 mL of Opti-MEM at a final concentration of 1 μM were added to the cells. The supernatants were collected after 20 or 8 h and stored at –80 °C until use. The level of TNF-α in the supernatants was determined by ELISA using OptEIA sets (BD, San Diego, CA, USA).

Human TNF-α and IL-6 were measured in culture supernatants using OptEIA sets (BD Bioscience, San Diego, CA USA). Culture supernatant was collected at 18 h for the detection of TNF-α, and at 48 h for IL-6.

Cytokine quantification in cell culture supernatants was performed by enzyme-linked immunosorbent assay (ELISA), according to the manufacturer’s instruction. The following cytokines were evaluated: IL-17, IL-6, TNF-α, IL-12p70, and IL-23 (DuoSets R&D Systems, Minneapolis, MN, USA) and IL-10 and IFN-γ (OptEIA sets, BD Biosciences, San Jose, CA, USA).