Serum free medium

Peripheral blood mononuclear cells (PBMCs) were isolated from healthy controls by Ficoll-Paque PREMIUM density gradient (GE Healthcare, USA) centrifugation. The CD14+ monocytes were separated by positive magnetic selection (Miltenyi Biotec, Germany). PBMCs were labeled with CD14 MicroBeads for 20 min at 4°C in the dark. Then, the cells were washed and immediately sorted on a MACS Separator (Miltenyi Biotec, Germany) to obtain CD14+ monocytes.
The CD14+ monocytes were cultured in serum-free RPMI-1640 medium for 2 h. After the cells adhered to the wall, the medium was changed to serum-free medium (LONZA, Switzerland) containing 20 ng/ml macrophage colony-stimulating factor (M-CSF) (PeproTech, USA). The medium was changed every 3 days and supplemented with M-CSF. The cells were induced to M0 macrophages (M0) after one week. To polarize into M1 macrophages (M1), cells were treated with 100 ng/ml LPS and 20 ng/ml interferon-γ (IFN-γ) (Novoprotein, Shanghai, China) for 24 h, and cells were stimulated with 20 ng/ml interleukin (IL)-4 (Novoprotein, Shanghai, China) for 24 h to polarize into M2 macrophages (M2).

IL-15:IL-15Rα, a heterodimer cytokine composed of IL-15 and IL-15Rα, was produced and purified from IL-15 and IL-15Rα transfected HEK293 cells. The cells were cultured in a serum-free medium (Lonza).

One or two cloacal swabs in stabilizing medium per flock with the highest C. gallinacea-DNA concentration(s) (Ct values 23.59–31.68) were chosen from six poultry flocks for isolation (Table 1) on Buffalo green monkey (BGM) cells. The isolation procedure was as described before [14 (link)] except that adjustments were made to the concentrations of nystatin (9 µg/mL), gentamycin (40 mg/mL), and vancomycin (25 mg/mL). A sample was considered positive when inclusions of typical chlamydial morphology appeared as bright apple-green spots upon staining with an IMAGEN Chlamydia kit (Oxoid Ltd., Basingstoke, UK) after two passages.
Additionally, strain 15-56/1 (Table 1) was propagated on BGM cell culture with UltraMDCK (Madin–Darby canine kidney) serum-free medium (Lonza, Cologne, Germany) in T25 flasks and incubated at 37 °C with 5% CO₂ in a fully humidified cabinet for 48–72 h [14 (link)]. The medium was replaced after 18 h.