Hi fetalplex

Mouse macrophage-like (J774A.1) cells (TIB-67; ATCC) and African green monkey kidney (Vero) cells (CCL-81; ATCC) were maintained in complete RPMI 1640 medium (i.e., RPMI 1640 without l-glutamine [Corning Cellgro; Corning Inc., Corning, NY], supplemented with GlutaMAX [Gibco BioSciences, Dublin, Ireland] and 5% [vol/vol] heat-inactivated serum complex [hi-FetalPlex; Gemini Bio-Products, West Sacramento, CA]) at 37°C and 5% CO₂. For infections, host cells were seeded in 12-well plates at a density of 2 × 10⁵ cells per 1.5 ml and maintained in complete RPMI 1640 at 37°C and 5% CO₂ for 24 h. Subsequently, host cells were infected with 1 × 10⁷ GE ml⁻¹ of bacteria in 1 ml of plain RPMI 1640 (i.e., RPMI 1640 without l-glutamine supplemented with only GlutaMAX). Infections were facilitated by centrifugation at 400 × g for 30 min at room temperature (RT). Following infection, inocula were removed and host cells were washed once with plain RPMI 1640 to remove noninternalized bacteria. Infected cells were maintained in 1.5 ml of RPMI 1640 (Corning Cellgro; Corning Inc., Corning, NY) containing 2% (vol/vol) hi-FetalPlex and incubated at 37°C and 5% CO₂.

The chemical composition of IFmM is shown in Table 1. Complete IFmM was prepared by mixing the base medium consisting of inorganic salts (pH 7.2), RPMI 1640 vitamins (Thermo Fisher Scientific, Waltham, MA), glucose, amino acids, and 5% (vol/vol) heat-inactivated serum complex (hiFetalPlex; Gemini Bio Products, West Sacramento, CA). Final pH was adjusted to 7.2 to 7.4 and the medium was filter sterilized (0.22 μm) and stored at 4°C.