Ultrapure 70 nitric acid

Overnight liquid cultures of H. pylori strain were grown until OD₆₀₀ 0.9 at 37°C in 6 ml Brucella broth (pH adjusted to 7), then 200 μM NiCl₂ were added and the cultures were left to grow until OD₆₀₀ 6. Then, the 6 ml of culture were centrifuged at 4000 g at 4°C for 25 min through 400 μL of a 1:2 mixture of the silicone oils AR20/AR200 (Wacker) in order to separate the cells from the medium. Cells were lysed with 400 μL 0.2 M NaOH/1% SDS for 60 min at 95°C. Samples were calibrated by protein concentration measurements with the DC Protein Assay kit (BioRad). Then, the samples were mineralized overnight at 80°C with 300 μL of ultrapure 70% nitric acid (JT Baker) and diluted to 1/20 in ultrapure water. Nickel contents were measured by ICP-OES using a Vista MPX spectrometer (Varian). The content of Ni(II) was determined using a curve established with certified ICP grade standards. The measurement of each strain in each condition was performed in triplicates in at least three independent experiments.

Overnight liquid cultures of H. pylori strain were grown until OD₆₀₀ 0.9 at 37°C in 6 ml Brucella-Broth with FCS, then 10 μM or 100 μM NiCl₂ were added and the cultures were left to grow until OD₆₀₀ 6. Then, the 6 ml of culture were centrifuged at 4000 g at 4°C for 25 min through 400 μL of a 1:2 mixture of the silicone oils AR20/AR200 (Wacker) in order to separate the cells from the medium. Cells were lysed with 400 μL 0.2 M NaOH/1% SDS for 60 min at 95°C. Samples were calibrated by protein concentration measurements with the DC Protein Assay kit (BioRad). Then, the samples were mineralized overnight at 80°C with 300 μL of ultrapure 70% nitric acid (JT Baker) and diluted to 1/20 in ultrapure water. Nickel contents were measured by ICP-OES using a Vista MPX spectrometer (Varian). The content of Ni(II) was determined using a curve established with certified ICP grade standards. The measurement of each strain in each condition was performed in triplicates in at least two independent experiments.