Heat killed listeria monocytogenes

The human leukemia monocytic cell line THP-1 (ATCC^®TIB-202) was cultured in RPMI-1640/L-glutamine (Biowest) enriched with 10% fetal calf serum (Thermo Fisher Scientific) and supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml) in a humidified environment at 5% CO₂ at 37°C. THP-1 monocytes were differentiated into macrophages by using 160 nM phorbol 12-myristate 13-acetate (PMA; Sigma Aldrich, Darmstadt, Germany) for 48 h on 0.24 × 10⁶ cells. S. saccharolyticus strains DVP5-16-4677 and 13 T0028, and S. aureus ATCC 25923 were cultured in BHCY broth to the mid-log growth phase. The bacteria were collected by centrifugation for 6 min at 5000 rpm, washed in sterile RPMI, resuspended, and diluted in RPMI to an OD₆₀₀ of 0.5. For preparing heat-killed bacteria, the bacterial suspensions were adjusted to 10⁸ CFU/ml; the bacterial suspensions were heat-killed at 90°C for 3 h. Two MOIs (multiplicity of infection) were used: MOI 10 and MOI 100. As a positive control, 10⁷ HKLM/mL (Heat-killed Listeria monocytogenes, Invivogen) was used.

Murine melanoma B16-F10 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and were cultivated in RPMI 1640 (Sigma-Aldrich, USA) supplemented with 10% fetal calf serum (FCS, PAA, Austria) and antibiotics. Cells were maintained at 37 °C in a humidified atmosphere with 5% carbon dioxide.
Heat killed Listeria monocytogenes was purchased from InvivoGen (Toulouse, France). SPF C57BL/6 mice (female, 18–20 g) were obtained from Charles River Laboratories (Sulzfeld, Germany). Mice were housed in barrier facilities with free access to sterile food and water. Photoperiod was 12/12, temperature 22 °C. All experimental mouse procedures were performed in accordance with the laws of the Czech Republic. Experimental project was approved by the Ministry of Education, Youth and Sports (protocol no. 28842/2014-3).