Columbia agar plates

The culture of Group B streptococci was performed as previously described.^{49 (link)} Briefly, the GBS strain BSU98 was freshly grown on Columbia agar plates (Sigma, Munich, Germany) supplemented with 5% defibrinated sheep blood and spectinomycin (150 g/ml; Sigma) for 16 h. Colonies were re-suspended in PBS and bacterial number was determined spectrometrically. Bacteria were inactivated for 30 min at 70 °C. For stimulation of PBMC and CBMC, inactivated GBS were used in a MOI of 1:50.

The culture of Group B streptococci was performed as previously described [25 (link)]. Briefly, the GBS strain BSU98 was freshly grown on Columbia agar plates (Sigma, Munich, Germany) supplemented with 5% defibrinated sheep blood, and spectinomycin (150 g/ml; Sigma) for 16 h. Colonies were re-suspended in PBS, and bacterial number was determined spectrometric. Bacteria were inactivated for 30 min at 70 °C. For stimulation of PBMC and CBMC with GBS, cells were seeded in 24 well plates (2 × 10⁶ cells/well) and stimulated with inactivated GBS in a multiplicity of infection (MOI) of 1:50.
A clinical isolate of E. coli K1 was grown on agar plates. After 16 h, a single colony was picked and grown in Lennox L broth-medium (Invitrogen, Karlsruhe, Germany) with or without supplements until early logarithmic growth. For stimulation of PBMC and CBMC, cells were seeded to 24 well plates (2 × 10⁶ cells/ml) and stimulated with E. coli in a MOI of 1:50.
For inhibition of TRIF, the Pepinh-TRIF inhibitor or the appropriate control peptide (both InvivoGen, France) were added during stimulation with E. coli.