1205 betaplate

3 × 10⁶ HeLa-hTDO cells were incubated with or without tetracycline (10 ng/mL) for 72 h under normoxia (20% O₂) or hypoxia (1% O₂) in 20 mL cell culture medium in culture flasks. Then the supernatants were harvested and used as cell culture medium for freshly isolated 1.5 × 10⁵ Ficoll-separated peripheral blood lymphocytes (PBL)/well. PBL were activated using the monoclonal anti-CD3 antibody OKT3; unstimulated PBL and tryptophan-supplemented PBL served as control group. T cell proliferation was determined after three days by adding ³H-thymidine for 24 h. The incorporation of ³H-thymidine was detected using liquid scintillation spectrometry (1205 Betaplate, PerkinElmer, Rodgau Jügesheim, Germany).

After pre-stimulation for 72 h, hRPE cells were infected with 2 × 10⁴T. gondii tachyzoites per well. T. gondii growth was determined by the ³H-uracil incorporation method as described before [26 (link), 29 (link)]. In brief, ³H-uracil (0.33 μCi per well) was added 48 h postinfection. Cultivation was stopped after additional 24 h by freezing. Parasite growth was determined by measuring incorporated ³H-uracil using liquid scintillation spectrometry (1205 Betaplate, PerkinElmer, Jugesheim, Germany).

1 × 10³ HeLa-hTDO cells were seeded per cm² in 25 cm² cell culture flask (Corning, NY, USA), ³H-thymidine was added at day 0, and the cells were incubated under normoxia (20% O₂) or hypoxia (1% O₂) for 1–7 days. The incorporation of ³H-thymidine was detected using liquid scintillation spectrometry (1205 Betaplate, PerkinElmer, Rodgau Jügesheim, Germany).

Peripheral blood mononuclear cells (PBMC) were prepared from the heparinised blood of infected and noninfected pigs after Ficoll density gradient centrifugation. For proliferation experiments, 1.5 × 10⁵ porcine PBMC per well were incubated in 200 μl Iscove's modified Dulbecco's medium (IMDM; Gibco, Grand Island, USA) containing 5% foetal calf serum (FCS) and 1% PenStrep (Biochrom, Berlin, Germany).
Cells were stimulated with toxoplasma lysate antigen (TLA) (10⁶ lysed RH T. gondii parasites per ml), recombinant antigens (1 μg/ml), concanavalin A (ConA; 1 μg/ml), or left untreated as negative control. After three to five days of incubation (dependent on the result of microscopic examination), 0.2 mCi [³H]-thymidine was added for 24 h and T cell proliferation was determined using liquid scintillation spectrometry (1205 Betaplate, PerkinElmer, Jügesheim, Germany).

Toxoplasma gondii (RH strain, ATCC, Wesel, Germany) or Neospora caninum (Nc-1 strain, kind gift of G. Schares, Greifswald-Insel Riems, Germany) tachyzoites were maintained in human foreskin fibroblasts (ATCC, Wesel, Germany) in IMDM containing 5% FCS. Tachyzoites were harvested after 5 days of incubation, resuspended in PBS, and counted. Preincubated HeLa-hTDO cells were infected with 3 × 10⁴ toxoplasma or 4 × 10⁴ neospora per well. Parasite growth was determined by the ³H-uracil incorporation method as described before [17 (link)]. In brief 48 h after infection 0.33 μCi ³H-uracil was added and after additional 24 h host cells were lysed by freeze and thaw cycles. The incorporation of ³H-uracil was detected using liquid scintillation spectrometry (1205 Betaplate, PerkinElmer, Rodgau Jügesheim, Germany).

After 72 h of stimulation with or without IFN-γ in the presence or absence of 1-MT or additional L-tryptophan, PCP-R cells were infected with 10⁵ T. gondii ME49 tachyzoites per well. The infected cells were incubated at 37°C in a 10% CO₂-enriched atmosphere. After 24 h, 0.012 MBq [³H]-uracil was added to the cells, and after lysis of the cells, the cell culture plates were frozen at −20°C. T. gondii proliferation was determined using liquid scintillation spectrometry (1205 Betaplate, PerkinElmer, Jügesheim, Germany).

1 × 10⁵ peripheral blood lymphocytes (PBL), obtained from heparinised blood of healthy donors after Ficoll purification, were stimulated with a monoclonal anti-CD3 antibody (OKT3, American Type Culture Collection, Rockville, USA) in the presence of different amounts of MSC as described [26 ]. In some experiments MSC (0.5–2 × 10⁴ per well) were infected with CMV and/or stimulated with IFN-γ at the start of the culture. After three days the cultures were pulsed with 0.2 μCi [³H] thymidine for 24 hours. T-cell proliferation was measured by [³H] thymidine incorporation using liquid scintillation spectrometry (1205 Betaplate, PerkinElmer, Jugesheim, Germany).