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Cell Meter™ Intracellular NADH/NADPH Flow Cytometric Analysis Kit *Red Fluorescence*

(A) Flow cytometric analysis of NADH/NADPH measurement in Jurkat cells using Cell Meter™ Intracellular NADH/NADPH Flow Cytometric Analysis Kit (Cat#15291). Cells were incubated with or without 100 µM NADH in serum-free medium for 30 minutes and then co-incubated with JZL1707 NAD(P)H sensor working solution for another 30 minutes.<br>(B) Fold increase of fluorescence signal intensity of Jurkat cells treated with 100 µM NADH or 100 µM NADPH compared<br>with untreated control. Fluorescence intensity was measured using ACEA NovoCyte flow cytometer in PE channel.
(A) Flow cytometric analysis of NADH/NADPH measurement in Jurkat cells using Cell Meter™ Intracellular NADH/NADPH Flow Cytometric Analysis Kit (Cat#15291). Cells were incubated with or without 100 µM NADH in serum-free medium for 30 minutes and then co-incubated with JZL1707 NAD(P)H sensor working solution for another 30 minutes.<br>(B) Fold increase of fluorescence signal intensity of Jurkat cells treated with 100 µM NADH or 100 µM NADPH compared<br>with untreated control. Fluorescence intensity was measured using ACEA NovoCyte flow cytometer in PE channel.
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Catalog Number15291
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Excitation (nm)535
Emission (nm)557
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


Excitation (nm)
535
Emission (nm)
557
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 Flow Cytometric Analysis Kit provides an efficient method to monitor intracellular NAD(P)H level in live cells. JZL1707 NAD(P)H sensor has been developed as an excellent fluorescent probe for detecting and imaging NADH/NADPH in cells. The probe bind NADH/NADPH to generate strong fluorescence signal with high sensitivity and specificity. JZL1707 NAD(P)H sensor can be readily loaded into live cells, and its fluorescence signal can be conveniently monitored using flow cytometer in PE channel.

Platform


Flow cytometer

Excitation488 nm laser
Emission575/26 nm filter
Instrument specification(s)PE channel

Components


Component A: JZL1707 NAD1 vial (100 uL)
Component B: Assay Buffer1 bottle (50 mL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells (0.5 - 1×106 cells/mL)
  2. Incubate cells with test compounds and JZL1707 NAD(P)H Sensor at 37 ºC for 30-60 minutes
  3. Wash and keep cells in Assay Buffer
  4. Analyze cells with a flow cytometer using PE channel

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

SAMPLE EXPERIMENTAL PROTOCOL

  1. For each sample, prepare cells in 0.5 mL serum-free medium or buffer of your choice at a density of 1×105 to 1×106 cells/mL. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density. For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intact, and wash the cells once with serum-free media. Note: JZL1707 NAD(P)H Sensor is serum sensitive, therefore it’s recommended to keep cells in serum-free medium or buffer of your choice. Alternatively, cells can be prepared and treated in regular full medium. Change to serum-free medium or buffer of your choice when incubation with JZL1707 NAD(P)H Sensor.                                                                                                                                                                                            
  2. Incubate cells with test compounds at 37 ºC for a desired period of time to stimulate intracellular NADH/NADPH. Note: The appropriate incubation time depends on the individual cell type and test compound used. Optimize the incubation time for each experiment.

  3. Add 1 µL of JZL1707 NAD(P)H Sensor (Component A) into 0.5 mL cell suspension. Incubate at 37 ºC for 30-60 minutes. Note: For a NADH/NADPH positive control treatment: Jurkat cells were incubated with 100 µM NADH or NADPH for 30 minutes in serum-free medium, and co-incubated with JZL1707 NAD(P)H Sensor working solution at 37 ºC for another 30 minutes. See Figure 1 for details.

  4. Wash cells with your desired buffer once. Keep cells in Assay Buffer (Component B).

  5. Monitor the fluorescence intensity at PE channel using a flow cytometer. Gate on the cells of interest, excluding debris.

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)535
Emission (nm)557

Citations


View all 58 citations: Citation Explorer
Activation of AhR-NQO1 signaling pathway protects against alcohol-induced liver injury by improving redox balance
Authors: Dong, Haibo and Hao, Liuyi and Zhang, Wenliang and Zhong, Wei and Guo, Wei and Yue, Ruichao and Sun, Xinguo and Zhou, Zhanxiang
Journal: Cellular and Molecular Gastroenterology and Hepatology (2021)
Dichloroacetate radiosensitizes hypoxic breast cancer cells
Authors: de Mey, Sven and Dufait, In{\`e}s and Jiang, Heng and Corbet, Cyril and Wang, Hui and Van De Gucht, Melissa and Kerkhove, Lisa and Law, Ka Lun and Vandenplas, Hugo and Gevaert, Thierry and others,
Journal: International journal of molecular sciences (2020): 9367
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

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