N8285

Manufactured by Merck Group

N8285 is a laboratory equipment product. It is a compact and versatile device designed for use in various scientific and research applications. The core function of N8285 is to provide precise and reliable measurements. Further details about its intended use or specific applications are not available.

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Lab products found in correlation

Nad nadh glo assay, by Promega (2 mentions) Phosphate buffered saline (pbs), by Merck Group (1 mentions) Clariostar microplate reader, by BMG Labtech (1 mentions)

2 protocols using n8285

Quantifying NAD+/NADH Ratio Assay

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NAD⁺/NADH was quantified using a NAD/NADH-Glo Assay (Promega, G9071) according to the manufacturer s instructions. A standard curve was established to ensure that signals were obtained within the linear range of detection. To do so, purified NAD⁺ or NADH (Sigma-Aldrich, N8285 or N6660, respectively) were quantified individually by performing tenfold serial dilutions according to the concentration ranges specified. Ratios of the two were then mixed at 4:1 and 1:4 to ensure the scaling factors were accurate. As NAD⁺ and NADH were not quantified separately in the experiments, our units are relative light units (RLU). All ratios (0–100%) of NAD⁺/NADH were quantifiable within the same linear RLU range (Supplementary Fig. 4c, right). Measurements were obtained after 45 min of incubation according to the manufacturer’s instructions. NAD⁺/NADH measurements were obtained from three biological replicates for all conditions at t₀.

Lopatkin A.J., Stokes J.M., Zheng E.J., Yang J.H., Takahashi M.K., You L, & Collins J.J. (2019). Bacterial metabolic state more accurately predicts antibiotic lethality than growth rate. Nature microbiology, 4(12), 2109-2117.

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Quantifying Cellular NAD+ Levels

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Changes in total cellular NAD⁺ levels after treatment with NMN were quantified using the NAD/NADH-Glo assay (#G9071, Promega) according to the manufacturer’s instructions. Standard curve for NAD⁺ was generated using defined concentrations of NAD⁺ prepared in PBS (#N8285, Sigma-Aldrich). Lysates were mixed with 0.4 N HCl, incubated at 60 °C for 15 min, cooled for 10 min at room temperature, and incubated with Trizma base prior to quantification. The Detection Reagent contained: Luciferin Detection Reagent, Reductase substrate, Reductase, Cycling Enzyme and NAD⁺ Cycling substrate. Samples and standards were mixed 1:1 with the Detection reagent prior to measuring luminescence on a luminometer. Sample luminescence values were normalized to protein concentration measured using the BioRad DC Protein assay kit on a CLARIOstar microplate reader (BMG Labtech).

Majeed Y., Halabi N., Madani A.Y., Engelke R., Bhagwat A.M., Abdesselem H., Agha M.V., Vakayil M., Courjaret R., Goswami N., Hamidane H.B., Elrayess M.A., Rafii A., Graumann J., Schmidt F, & Mazloum N.A. (2021). SIRT1 promotes lipid metabolism and mitochondrial biogenesis in adipocytes and coordinates adipogenesis by targeting key enzymatic pathways. Scientific Reports, 11, 8177.

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