HEK293T cells (6 × 106) were seeded in 100 mm plates a day before the experiment. Cells were ~ 80% confluent at the time of protein extraction. To measure cellular total NAD levels, 1/5 of the cells were lysed in 400 μl of NAD/NADH Extraction Buffer (NAD/NADH Quantitation Kit, Sigma-Aldrich) and total protein was extracted by two freeze/thaw cycles (20 min on dry –ice, then 15 min at RT). Cell lysates were centrifuged at 13,000g at 4°C for 10 min and supernatant was taken to measure protein concentration. 10 μg of total proteins were used to detect cellular NAD according to the manufacture’s protocol (NAD/NADH Quantitation Kit, Sigma).
Nad nadh quantitation kit
Manufactured by Merck Group
Sourced in United States, Germany
The NAD/NADH Quantitation Kit is a laboratory equipment product designed to quantify the levels of NAD (Nicotinamide Adenine Dinucleotide) and NADH (Nicotinamide Adenine Dinucleotide, Reduced) in biological samples. The kit provides the necessary reagents and protocols to perform colorimetric or fluorometric measurements, allowing for the determination of NAD and NADH concentrations.
Automatically generated - may contain errors
Lab products found in correlation
Nuclease p1, by Merck Group (3 mentions)
Fk866, by Merck Group (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Opti mem medium, by Thermo Fisher Scientific (2 mentions)
Acetyl lysine antibody, by Cell Signaling Technology (2 mentions)
Anti ezh2, by Cell Signaling Technology (2 mentions)
Gc fid, by Hewlett-Packard (1 mentions)
Hp1047a, by Agilent Technologies (1 mentions)
Agilent 1100 series hplc system, by Agilent Technologies (1 mentions)
U0126, by Merck Group (1 mentions)
Gsk1120212, by Euromedex (1 mentions)
Pd98059, by Selleck Chemicals (1 mentions)
Cas 285986 31 4, by Merck Group (1 mentions)
Hpx 87h column, by Aminex (1 mentions)
Uhplc system, by Agilent Technologies (1 mentions)
Nadp nadph quantitation kit, by Merck Group (1 mentions)
Uplc beh amide column, by Waters Corporation (1 mentions)
Tripletof 6600 q tof, by AB Sciex (1 mentions)
Pyruvate, by Merck Group (1 mentions)
Spectramax m2e, by Molecular Devices (1 mentions)
39 protocols using nad nadh quantitation kit
1
Cellular NAD Levels Quantification
2
Quantification of Cellular NAD Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Yeast strains were grown in YPD media at 30 °C according to standard protocols. All strains were harvested for the experiments at exponential phase (OD600 ~1) and total RNA was isolated using the acidic hot phenol method48 (link). To remove residual free NAD, purified RNAs were dissolved in 10 mM Tris-HCl (pH 7.5) containing 2 M urea. Samples were incubated 2 min at 65 °C and immediately precipitated with isopropanol in the presence of 2 M ammonium acetate. NAD-capQ was carried out as previously described21 (link). Briefly, 50 μg of total RNA was digested with 2 U of Nuclease P1 (Sigma-Aldrich) in 20 μL of 10 mM Tris (pH 7.0), 20 μM ZnCl2 at 37 °C for 1 h to release 5′-end NAD. The control samples lacking Nuclease P1 were prepared by incubating 50 μg of RNA treated with the same reaction condition. The NADH standard curve was generated for each experiment in the same buffer condition as above for assays containing Nuclease P1. Following digestion with Nuclease P1, 30 μL of NAD/NADH Extraction Buffer (NAD/NADH Quantitation Kit, Sigma-Aldrich) was added to each sample. In the second step, 50 μL samples were used in a colorimetric assay according to the manufacturer’s protocol (NAD/NADH Quantitation Kit, Sigma-Aldrich) as described21 (link).
3
Quantification of NAD and NADH in HEK293T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T cells (6 × 106) were seeded in 100 mm plates a day before the experiment and cells were collected for RNA extraction at ∼80% confluency. NAD-capQ was carried out as previously described (15 (link)) (Figure 2A ). Briefly, 50 μg of total RNA was digested with 2 U of Nuclease P1 (Sigma-Aldrich) in 20 μl of 10 mM Tris (pH 7.0), 20 μM ZnCl2 at 37°C for 1 h to release 5′ end NAD. The control samples lacking Nuclease P1 were prepared by incubating 50 μg of RNA treated with the same reaction condition and supplemented with 5% glycerol lacking the enzyme. The NADH standard curve was generated for each experiment in the same buffer condition as above for assays containing Nuclease P1. Following digestion with Nuclease P1, 30 μl of NAD/NADH Extraction Buffer (NAD/NADH Quantitation Kit, Sigma-Aldrich) was added to each sample. Fifty microliters of each sample was used in a colorimetric assay according to the manufacturer's protocol (NAD/NADH Quantitation Kit, Sigma-Aldrich) as described (15 (link)). Values were corrected for background absorbance and concentrations of NAD and NADH were derived from the standard curves.
4
NAD+/NADH Quantification from Cell Extracts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The extraction was done aerobically. Cells were lysed in 600 μL extraction buffer from the NAD+/NADH Quantitation Kit (Sigma-Aldrich, Darmstadt, Germany) and 300 μL chloroform supplemented with 400 mg glass beads in a Mixer Mill MM 400 (Retsch GmbH, Haan, Germany). The procedure included three cycles of homogenization with 20 seconds break in between at < -20°C. After thawing, the samples were centrifuged (5 min, 16,000 x g, 4°C). Quantification of NAD+/NADH was conducted with the NAD+/NADH Quantitation Kit obtained from Sigma (Sigma-Aldrich, Darmstadt, Germany) according to the supplier’s manual.
5
Cellular NAD Levels Quantification
6
Quantification of Cellular NAD Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Yeast strains were grown in YPD media at 30 °C according to standard protocols. All strains were harvested for the experiments at exponential phase (OD600 ~1) and total RNA was isolated using the acidic hot phenol method 41 . To remove residual free NAD, purified RNAs were dissolved in 10 mM Tris-HCl (pH 7.5) containing 2 M urea. Samples were incubated 2 min at 65 °C and immediately precipitated with isopropanol in the presence of 2 M ammonium acetate. NAD-capQ was carried out as previously described 21 (link) . Briefly, 50 µg of total RNA was digested with 2 U of Nuclease P1 (Sigma-Aldrich) in 20 µL of 10 mM Tris (pH 7.0), 20 µM ZnCl2 at 37 °C for 1h to release 5′-end NAD. The control samples lacking Nuclease P1 were prepared by incubating 50 µg of RNA treated with the same reaction condition. The NADH standard curve was generated for each experiment in the same buffer condition as above for assays containing Nuclease P1. Following digestion with Nuclease P1, 30 µL of NAD/NADH Extraction Buffer (NAD/NADH Quantitation Kit, Sigma-Aldrich) was added to each sample. In the second step, 50 µL samples were used in a colorimetric assay according to the manufacturer's protocol (NAD/NADH Quantitation Kit, Sigma-Aldrich) as described 21 (link) .
7
Exosome-Induced Metabolic Changes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following cisplatin treatment, cells cocultured with exosomes were lysed using ultrasound. Nicotinamide adenine dinucleotide (NADH) and glutathione (GSH) levels were measured using the NAD/NADH Quantitation Kit (MAK037, Sigma-Aldrich, USA) and Reductive GSH Content Assay Kit (Solarbio, Beijing, China) respectively, based on instructions provided by the manufacturer. Reactive oxygen species (ROS) levels were measured using a fluorescent 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) assay as described by the manufacturers (Jiancheng, Nanjing, China). All data were normalized to total cell number.
8
Metabolic Analysis of 2,3-Butanediol Fermentation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell growth was monitored by measuring optical density at 600 nm (OD600). Glucose was determined using a glucose analyzer. Extracellular metabolite concentrations were determined by HPLC [33 (link)]. The mobile phase consisted of 5 mM H2SO4 with the flow rate at 0.4 ml/min, and the column temperature was set at 65 °C. To determine the enantiomeric distribution of 2,3-BD, the fermentation samples were saturated with sodium chloride and extracted with ethyl acetate. The isomers in the extracts were then analyzed by GC-FID (Hewlett Packard) equipped with a HP-chiral 20b column as described previously [57 (link)]. The oven temperature program was as follows: 40 °C (2 min), increased to 75 °C (4 min) at 5 °C min−1, followed by a ramp of 1 °C min−1 to 80 °C (2 min), and finally, 15 °C min−1 to 230 °C (4 min). The intracellular NADH and NAD+ levels were determined by NAD/NADH Quantitation Kit (SIGMA).
9
Quantifying NADH and Total NAD
10
Monitoring Cell Growth and Metabolite Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell growth was monitored by measuring the OD600 with an ultraviolet spectrophotometer (Beijing Puxi Universal Co. Ltd). The biomass concentration was calculated from OD600 values using an experimentally determined correlation with 1 OD600 unit being equal to 0.38 g/L cell dry weight (CDW) [42 (link)]. Glucose in the fermentation broth was determined utilizing a SBA sensor machine (Institute of Microbiology, Shangdong, China). To determine succinate, pyruvate, acetate and lactate concentrations, culture samples were centrifuged at 12,000g for 5 min and the aqueous supernatant used for HPLC analysis on an Agilent 1100 Series HPLC system equipped with a cation exchange column. (Aminex HPX87-H, Bio-Rad, Hercules, CA, USA), a UV absorbance detector (Agilent Technologies, G1315D) and a refractive index (RI) detector (Agilent Technologies, HP1047A). A mobile phase of 5 mM H2SO4 solution at a 0.4 mL/min flow rate was used. The column was operated at 65 °C. The intracellular NADH and NAD+ levels were determined by NAD/NADH Quantitation Kit (SIGMA) [47 (link)].
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!