MPOe Activity Quantification

To determine the activation of BMDNs infected with each bacterial strain separately we measured the enzymatic activity of MPO using the Neutrophil Myeloperoxidase Activity Assay Kit (Cayman Chemical). Briefly, infected BMDNs were incubated and at 1 and 3 hpi the color intensity of the 3,3′,5,5′-tetramethyl-benzidine (TMB), which is proportional to the amount of MPO in the sample and is detectable at 650 nm, obtaining the enzymatic activity, in μmoles/min./ml. To obtain the enzymatic specific activity, the results were normalized to the total protein concentration in the samples. Negative control of non-infected neutrophils and free bacteria were used for normalization, in addition to the negative controls with MPO inhibitor (4-aminobenzhydrazide) provided by the kit.