Nadp nadph fluorometric assay kit

Cells were disrupted in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA pH 8.0, 0.5% TritonX-100, complete Mini protease inhibitors [Roche; Indianapolis, IN], 100 μM phenylmethylsulfonyl fluoride (PSMF), and 100 μM sodium orthovanadate). Cell debris was removed by centrifugation at 12,000 × g and 4°C for 15 minutes. The supernatant fraction was collected, and protein concentration was determined at 590 nm using Bio-Rad Bradford Protein Assay (Biorad). NADP/NADPH fluorometric assay kit (Abcam) was used as directed by the manufacturer to determine intracellular ratios of these metabolites. 590 nm absorbance was read with a VictorX5 96-well plate reader (Perkin-Elmer).

The levels of NADP and NADPH produced during Spm stress were measured using the NADP/NADPH fluorometric assay kit (Abcam) according to the manufacturer’s instructions, with slight modifications as follows. Bacteria cultured to the logarithmic phase were diluted to an OD₆₀₀ of 0.2. After centrifugation, the collected bacterial pellet was resuspended in an equal volume of the lysis buffer. These were incubated for at least 1 hour on a heating block [37°C, with agitation (200 rpm)]. Cell lysates were collected by centrifugation, and supernatants were stored at −20°C or used directly for NADP/NAPH level quantification according to the manufacturer’s instructions.
For the quantification of the levels of ferric (Fe³⁺) iron and ferrous (Fe²⁺) iron produced during Spm stress, the culture OD₆₀₀ was between 0.8 and 1 since lower optical densities were below the detection limit. A volume of 100 µL of either the Spm-treated sample or the control was aliquoted in a 96-well plate. Then, ferric iron and ferrous iron were quantified using an iron assay kit (Sigma-Aldrich Co. LLC) following the manufacturer’s instructions.