Phytohemagglutinin l pha

Our IFN-γ ELISpot protocol is described in detail elsewhere [2 (link),22 (link)]. The ELISpot was performed in technical duplicates. Here, 50,000 cells/well were seeded for the virus-derived peptide stimulation and 300,000 cells/well for the background assessment (10% DMSO, negative control). Phytohemagglutinin-L (PHA, 10 µg/mL, Sigma-Aldrich) was used as a positive control. Spots were counted with the ImmunoSpot series 6 ultra-V analyzer (CTL Europe GmbH) according to the laboratory standard protocol. No cut-off was set for the spot count.

1 × 10⁵ MNCs were seeded into 96-well U bottom plates (Greiner bio-one, Frickenhausen, Germany) in RPMI medium supplemented as above with or without 6 nmol/mL each of Peptivator HPV16 E6 & E7 (Miltenyi Biotec, Bergisch–Gladbach, Germany) for 24 h. The assay was then transferred to IFN-γ-coated MultiScreen-HTS-IP (Merck Millipore, Darmstadt, Germany) for another 48 h. IFN-γ capture (clone 1-D1K) and biotinylated detection (clone 7-B6-1) antibodies were purchased from Mabtech AB (Cologne, Germany); ExtrAvidin®–Alkaline Phosphatase and BCIP/NBT solution was purchased from Sigma-Aldrich (Taufkirchen, Germany). Spots were counted by an AID iSpot FluoroSpot Reader System with the EliSpot 6.0 iSpot software (AID Diagnostika, Straßberg, Germany). Phytohemagglutinin-L (PHA, Sigma-Aldrich, Taufkirchen, Germany) was utilized as a positive control.

Histopaque-1077, Dulbecco’s phosphate buffered saline (DPBS), Roswell Park Memorial Institute medium (RPMI 1640), Fetal calf serum (FCS), Dimethylsulfoxide (DMSO), Penicillin/Streptomycin, 2-mercaptoethanol and Trypan blue 0.4% were all purchased from ThermoFisher (France). Phorbol 12-myristate 13-acetate (PMA) ionomycin calcium salt, Phytohemagglutinin-L (PHA) and Concanavalin A (ConA) were purchased from Sigma-Aldrich (France). Toxoplasma serological test (TOXOPLASMA ICT IgG-IgM) was purchased from LD Bio Diagnostic (France). ELISPOT plates were purchased from Mabtech (Sweden). MicroBCA kit was purchased from Pierce (France). Vero cells were obtained from ATCC (CCL-81), as well as T.gondii tachyzoites (RH strain, ATCC 50174), and stored in liquid nitrogen until use.

Human PBMC were isolated from healthy donors using Lymphoprep density centrifugation. The cells were cultured in RPMI-1640 medium supplemented with 10% FCS, 10 mM of L-glutamine, Penicillin (100 U/ml)/Streptomycin (100µg/ml) and 25 mmol/L HEPES (all from Thermo Scientific HyClone, USA). PBMC were seeded at 2×10⁵ cells/well and stimulated at 37°C with serial dilutions of bacterial supernatants. After 72 hours, the cells were pulsed for 6 hours with 1 µCi/well of 3H-thymidine (Perkin-Elmer) after which 3H-uptake was measured in a beta-scintillation counter. Phytohemagglutinin-L (PHA) (1 µg/ml) (Sigma-Aldrich, St. Louis, USA) was used as a positive control for polyclonal T cell activation. The cytotoxic/inhibitory effect was tested by the addition of bacterial supernatants in combination with PHA in the proliferation assay. The bacterial culture medium CCY was included as a negative control, and was found to have negligible effect on proliferation (mean CPM 3088, 1785, 1719 for 1∶50; 1∶100 and 1∶1000 dilution, resp.) and no inhibitory effect on PHA-induced proliferation. PBMC were also stimulated with bacterial supernatants or PHA in combination with purified α-toxin (Sigma-Aldrich, St. Louis, USA), recombinant PVL, recombinant δ-toxin and purified PSM-α3 (all from IBT Bioservices, Gaithersburg, USA) and proliferation assessed.