Elisa assays

For quantitative bacterial analysis, blood (25μl) was spread directly onto blood agar plates (Oxoid, Hampshire, UK), incubated at 37°C and bacterial colonies were enumerated at 24-48h. In parallel, macrophage inflammatory protein 2 (MIP-2), keratinocyte chemoattractant (KC), tumor necrosis factor (TNF)-α and interleukin (IL)-1β, the pathogenesis associated cytokines of C. difficile infection [2 (link)], were measured in serum with ELISA assays (PeproTech, NJ, USA). All assays were performed according to the manufacturer’s protocol. For the fungal burden in blood, blood (50 μl), in serial dilution, was directly plated on Sabouraud Dextrose Agar (SDA) with 0.1% chloramphenicol (Thermo Scientific, Hampshire, UK) and Sabouraud Dextrose Broth (SDB) with 0.05 g/l chloramphenicol (Thermo Scientific), then kept at 35°C for 72h before Candida colony enumeration. In addition, the colons of the mice were collected at sacrifice, weighed, homogenized with PBS and used for tissue cytokine analysis (MIP-2, KC, TNF-α and IL-1β) as biomarkers of local inflammatory responses.

For quantification of growth factors and protein content in the cells, mononuclear cells from control and both BMAC groups, after Biocoll separation, were lysed (3 freeze/thaw cycles), and the lysate supernatants were then analyzed using the Quantibody-Array QAH-BMA-1000-2 (RayBiotech, Norcross, GA, USA) and ELISA assays (Peprotech, Hamburg, Germany) following the manufacturer’s instructions.

Venous blood samples were collected and centrifuged at 1000 rpm for 5 minutes. Plasma from the top of the tubes was withdrawn and centrifuged again to obtain clear plasma. Samples (220 μl) were stored in a freezer at −80°C until analysis. Cytokine levels of TNF-α and IL-6 were performed in duplicate by commercially available immunoenzymatic (ELISA) assays (BioSource International, Inc., Ca, USA). C-reactive protein (CRP) was quantified in duplicate using ultrasound kits on Mindray BS-200 automated biochemical equipment.