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Cell Meter™ Intracellular NADH/NADPH Fluorescence Imaging Kit *Deep Red Fluorescence*

Fluorescence images of NADH/NADPH in HeLa cells using Cell Meter™ Intracellular NADH/NADPH Fluorescence Imaging Kit (Cat#15295). HeLa cells were incubated with 100 µM NADH or 100 µM NADPH in serum-free medium for 30 minutes and then co-incubated with JJ1902 NAD(P)H sensor working solution for another 30 minutes. The fluorescence signal was measured using fluorescence microscope with a Cy5® filter.
Fluorescence images of NADH/NADPH in HeLa cells using Cell Meter™ Intracellular NADH/NADPH Fluorescence Imaging Kit (Cat#15295). HeLa cells were incubated with 100 µM NADH or 100 µM NADPH in serum-free medium for 30 minutes and then co-incubated with JJ1902 NAD(P)H sensor working solution for another 30 minutes. The fluorescence signal was measured using fluorescence microscope with a Cy5® filter.
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Catalog Number15295
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


The detection of intracellular dihydronicotinamide adenine dinucleotide NADH and its phosphate ester NADPH is important for disease diagnostics and drug discovery. In general, the redox couples NAD/NADH and NADP/NADPH play a critical role in energy metabolism, glycolysis, tricarboxylic acid cycle and mitochondrial respiration. The increased NAD(P)H level in cells is linked to the abnormal production of reactive oxygen species (ROS) and DNA damage. However, due to the lack of sensitive NAD(P)H probe, it has been challenging to detect intracellular NAD(P)H in biological systems. Cell Meter™ Intracellular NADH/NADPH Fluorescence Imaging Kit provides an efficient method to monitor intracellular NAD(P)H level in live cells in the far spectrum and can be combined with other applications such as GFP-expressed cells or application of MitoTracker. JJ1902 NAD(P)H sensor has been developed as an excellent fluorescent probe for detecting and imaging NADH/NADPH in cells. The probe which is fluorogenic in nature, binds NADH/NADPH to generate strong fluorescence signal with high sensitivity and specificity. JJ1902 NAD(P)H sensor can be readily loaded into live cells, and its fluorescence signal can be conveniently monitored using the filter set of Cy5®. This kit is optimized for fluorescence imaging and microplate reader applications.

Platform


Fluorescence microscope

Excitation590 nm
Emission655 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Cy5 filter

Components


Component A: JJ1902 NAD(P)H Sensor1 vial (40 µL)
Component B: Assay Buffer1 bottle (20 mL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells in growth medium
  2. Incubate cells with test compounds and JJ1902 NAD(P)H Sensor working solution at 37 oC 20 - 30 minutes 
  3. Wash and keep cells in Assay Buffer
  4. Monitor fluorescence intensity (bottom read mode) at Ex/Em = 590/655 nm (Cutoff = 610 nm) or fluorescence microscope with Cy5® filter

Important
Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF WORKING SOLUTION

Add 10 µL of JJ1902 NAD(P)H Sensor stock solution (Component A) into 2.5 mL of Assay Buffer (Component B), and mix well. This JZL1707 NAD(P)H Sensor working solution is stable within 1 hour at room temperature. Note: 40 µL of JJ1902 NAD(P)H Sensor stock solution is enough for one plate. Protect from light.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

  1. To stimulate NADP/NADPH, treat cells with 10 µL of 10X test compounds (96-well plate) or 5 µL of 5X test compounds (384-well plate) in serum free medium or your desired buffer (such as PBS or HHBS). For control wells (untreated cells), add the corresponding amount of medium or compound buffer. Note: JJ1902 NAD(P)H sensor is compatible in the presence of serum as well. The optimization of the conditions for the sensor is highly recommended cell line to cell line.                                                                                                                                                                                    
  2. Add 100 µL/well (96-well plate) or 25µL/well (384-well plate) of JJ1902 NAD(P)H Sensor working solution in the cell plate. Co-incubate cells with test compound and JJ1902 NAD(P)H Sensor working solution at 37 oC for 20-30 minutes, protected from light. Note: For a NADH/NADPH positive control treatment: HeLa cells were incubated with 100 µM NADH or NADPH for 30 minutes in serum-free medium, and co-incubated with JJ1902 NAD(P)H sensor working solution at 37 oC for another 30 minutes. See Figure 1 for details.

  3. Wash cells with your desired buffer once. Remove solution in each well and add Assay Buffer (Component B) 100 µL/well for a 96-well plate or 25 µL/well for a 384-well plate.

  4. Monitor the fluorescence increase using microplate reader at Ex/Em = 590/655 nm (Cutoff = 610 nm) with bottom read mode, OR take images using fluorescence microscope with the filter set of Cy5® filter.

Citations


View all 56 citations: Citation Explorer
Resveratrol attenuates excessive ethanol exposure induced insulin resistance in rats via improving NAD+/NADH ratio
Authors: Luo, Gang and Huang, Bingqing and Qiu, Xiang and Xiao, Lin and Wang, Ning and Gao, Qin and Yang, Wei and Hao, Liping
Journal: Molecular Nutrition & Food Research (2017)
Epigenetic regulation of Runx2 transcription and osteoblast differentiation by nicotinamide phosphoribosyltransferase
Authors: Ling, Min and Huang, Peixin and Islam, Shamima and Heruth, Daniel P and Li, Xuanan and Zhang, Li Qin and Li, Ding-You and Hu, Zhaohui and Ye, Shui Qing
Journal: Cell & Bioscience (2017): 27
MCU-dependent mitochondrial Ca2+ inhibits NAD+/SIRT3/SOD2 pathway to promote ROS production and metastasis of HCC cells
Authors: Ren, T and Zhang, H and Wang, J and Zhu, J and Jin, M and Wu, Y and Guo, X and Ji, L and Huang, Q and Yang, H and others, undefined
Journal: Oncogene (2017)
Metabolic and molecular insights into an essential role of nicotinamide phosphoribosyltransferase
Authors: Zhang, Li Q and Van Ha, undefined and el, Leon and Xiong, Min and Huang, Peixin and Heruth, Daniel P and Bi, Charlie and Gaedigk, Roger and Jiang, Xun and Li, Ding-You and Wyckoff, Gerald and others, undefined
Journal: Cell Death & Disease (2017): e2705
Cytosolic Redox Status of Wine Yeast (Saccharomyces Cerevisiae) under Hyperosmotic Stress during Icewine Fermentation
Authors: Yang, Fei and Heit, Caitlin and Inglis, Debra L
Journal: Fermentation (2017): 61
Celastrol attenuates angiotensin II mediated human umbilical vein endothelial cells damage through activation of Nrf2/ERK1/2/Nox2 signal pathway
Authors: Li, Miao and Liu, Xin and He, Yongpeng and Zheng, Qingyin and Wang, Min and Wu, Yu and Zhang, Yuanpeng and Wang, Chaoyun
Journal: European Journal of Pharmacology (2017): 124--133
Pyrroloquinoline Quinone, a Redox-active o-Quinone, Stimulates Mitochondrial Biogenesis by Activating SIRT1/PGC-1α Signaling Pathway
Authors: Saihara, Kazuhiro and Kamikubo, Ryosuke and Ikemoto, Kazuto and Uchida, Koji and Akagawa, Mitsugu
Journal: Biochemistry (2017)
Engineering a glycerol utilization pathway in Corynebacterium glutamicum for succinate production under O2 deprivation
Authors: Wang, Chen and Cai, Heng and Chen, Zhongjun and Zhou, Zhihui
Journal: Biotechnology letters (2016): 1791--1797
Efficient testosterone production by engineered Pichia pastoris co-expressing human 17β-hydroxysteroid dehydrogenase type 3 and Saccharomyces cerevisiae glucose 6-phosphate dehydrogenase with NADPH regeneration
Authors: Shao, Minglong and Zhang, Xian and Rao, Zhiming and Xu, Meijuan and Yang, Taowei and Li, Hui and Xu, Zhenghong and Yang, Shangtian
Journal: Green Chemistry (2016): 1774--1784
Impact of Genetic Reduction of NMNAT2 on Chemotherapy-Induced Losses in Cell Viability In Vitro and Peripheral Neuropathy In Vivo
Authors: Slivicki, Richard A and Ali, Yousuf O and Lu, Hui-Chen and Hohmann, Andrea G
Journal: PloS one (2016): e0147620

References


View all 1 references: Citation Explorer
Inhibition of leucine aminopeptidase 3 suppresses invasion of ovarian cancer cells through down-regulation of fascin and MMP-2/9
Authors: Wang X, Shi L, Deng Y, Qu M, Mao S, Xu L, Xu W, Fang C.
Journal: Eur J Pharmacol (2015): 116