Macsquant analyser

Spleen cells were incubated with anti-mouse CD16/CD32 mAb (2.4G2; BD Biosciences, San Jose, CA) in FACS buffer (HBSS containing 2% FCS) for FcR blocking for 15 min on ice, and then incubated with BV421-conjugated anti-mouse CD3 mAb (145-2C11; Biolegend, San Diego, CA), APC-conjugated anti-mouse CD8 mAb (53-6.7; Biolegend), PE-conjugated anti-mouse CD4 (GK1.5 Biolegend) or Siglec F (E50-2440; BD Biosciences) mAb, PE-Cy7-conjugated anti-mouse CD19 mAb (6D5; Biolegend) or Gr1 (RB6-8C5; Affymetrix eBioscience, San Diego, CA) mAb for 20 min on ice. The expression profile of each cell surface marker on 7-aminoactinomycin D (7-AAD; Sigma-Aldrich Co. LLC, St. Louis, MO) -negative cells was analyzed on a MACSQuant Analyser (Miltenyi Biotec, Bergisch Gladbach, Germany) with MACSQuantify Software (Miltenyi Biotec) and FlowJo software (Tree Star Inc., Ashland, OR).

For this assay, 2 × 10⁴ HCT 116 were seeded in a 96-well cell culture plate and incubated for 24 h at 37 °C. The culture medium was replaced with 100 µl medium containing sc1o in various concentrations or control substances. As a negative control, medium with 10% FCS was used. G1/S-block was induced via medium without 10% FCS. Twenty µM curcumin and 0.2 µM staurosporine were used to induce G2- and S-block, respectively. After an incubation step of 24 h at 37 °C, cells were harvested, suspended in 200 µl sample buffer (1 g glucose/1 l phosphate-buffered saline (PBS) without calcium or magnesium), mixed, centrifuged (200 g, 4 min, 4 °C), and the supernatant was discarded. This step was repeated once. Cells were fixed with 150 µl of ice-cold 70% ethanol overnight (>18 h) at 4 °C. Cell pellet was washed with sample buffer, resuspended in 100 µl staining buffer (20 µ/ml propidium iodide and 0.2 mg/ml RNase in sample buffer) and incubated for 40 min at RT. Samples were measured within 24 h in a MACSQuant analyser (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Cell cycle distribution was determined using FlowJo software.

Spleens were taken at autopsy and strained through a 100-μm cell strainer (Fisher Scientific, Loughborough, UK), and red blood cells were removed by treatment with a red blood cell lysis buffer (Sigma Aldrich, MO, USA). Cells from T. gondii-infected mice were plated at 1 × 10⁶ cells/well and incubated in the presence of STAg (soluble tachyzoite antigen) (25 μg/ml) for 24 h or ConA (2.5 μg/ml) for 72 h. STAg was produced from cultured tachyzoites, freeze thawed in liquid nitrogen and filtered.
Single-cell suspensions were prepared from mesenteric lymph nodes (MLNs) from T. muris-infected mice and plated at 1 × 10⁶ cells/well of a 96-well plate. Cells were incubated with E/S antigen (50 μg/ml) for 48 h or ConA (2.5 μg/ml) for 72 h. All cells were incubated at 37 °C, 5% CO₂ and after incubation supernatants were harvested and stored at −80 °C. Levels of IL-10, 1 L-13, IL-4, IL-6, IL-1β, IL-1α, interferon gamma (IFN-γ), TNF-α, CXCL-1, CCL5 and CCL2 were determined using a custom cytometric bead array (CBA) according to the manufacturer’s instructions (BD Biosciences, Oxford, UK), and plates were read using a MACSQuant Analyser (Miltenyi Biotec, Cologne, Germany). Analysis was carried out using FCAP Array™ Software (BD Biosciences).