Protein carbonyl colorimetric

Plasma levels of 25(OH)vitamin D were determined using an ELISA kit (Calbiotech, Spring Valley, CA), levels of insulin were determined using ELISA kits from ALPCO Diagnostics (Salem, NH); the HOMA insulin resistance index was calculated^{48 (link)}. Protocols, as provided in the manufacturer’s instructions, were followed, including the use of appropriate controls and standards. Blood glucose was assessed using an Accu-Chek glucometer (Boehringer Mannheim Corp., Indianapolis, IN) with blood obtained via tail prick. Levels of GSH in plasma and tissues were determined using HPLC^{47 (link)}, using an assay that determines total GSH status. GSH levels in cultured cells were quantified using a fluorimetric method (CS1020; Sigma, Saint Louis, Missouri) Oxidative stress was assessed by the quantification of protein carbonyls and MDA using Protein Carbonyl Colorimetric and TBARS Assay Kits, respectively (Cayman Chemical, Ann Arbor, MI).

As indicators of oxidative damage, we determined protein carbonylation, lipid peroxidation, and total glutathione using the Protein Carbonyl Colorimetric, TBARS, and Glutathione Assay Kits (Cayman Chemical), respectively. Briefly, we infected BMDNs with S. Typhimurium 14028s and ΔarcA separately, as described above. For protein carbonylation, we measured the absorbance at a wavelength of 375 nm of the hydrazone formed by the reaction between 2,4-dinitrophenylhydrazine and the target molecule to determine the concentration (nmol/ml) of protein carbonyls. For TBARS, we used an excitation wavelength of 530 nm and an emission wavelength of 550 nm, and lipid peroxidation was determined as the malondialdehyde concentration (μM) obtained. For glutathione, we used a 410 nm wavelength to read the microplate and obtain the rate of 5-thio-2-nitrobenzoic acid production, which is directly proportional to the concentration of total glutathione (μM). Measurements for each indicator were performed at 1 and 3 hpi. In all cases, negative controls of non-infected neutrophils and free bacteria were used for normalization.