Cell Meter™ Live Cell ATP Assay Kit

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R-phrase	R20, R21, R22
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Overview SDS Protocol

Adenosine triphosphate (ATP) plays a fundamental role in cellular energetics, metabolic regulation and cellular signaling. It is referred as the "molecular unit of currency" of intracellular energy transfer to drive many biological processes and chemical synthesis in living cells. ATP also serves as a signaling molecule for cell communication and plays an important role in DNA and RNA synthesis. It is localized in mitochondria, where cellular respiration occurs. ATP levels can be used to measure cell proliferation and cell cycle dynamics. AAT Bioquest offers a variety of bioluminescence, fluorescence and colorimetric assay kits to determine ATP level in solutions. Cell Meter™ Live Cell ATP Assay Kit enables researchers to monitor ATP levels in live cells using ATP Red™, a cell-permeable red fluorescent imaging probe for detecting ATP. ATP Red™ is designed to monitor ATP concentrations in the mitochondria of living cells. The probe has minimal cross reactivity to AMP, ADP, CMP, CDP, CTP, UMP, UDP, UTP, GMP, GDP or GTP.

Platform

Fluorescence microscope

Excitation	Cy3/TRITC filter set
Emission	Cy3/TRITC filter set
Recommended plate	Black wall/clear bottom

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare cells in growth medium
Incubate cells with ATP Red™ working solution at 37 °C for 15-30 minutes
Remove ATP Red™ working solution
Analyze with a fluorescence microscope using a Cy3/TRITC filter set

CELL PREPARATION

For adherent cells

Plate cells overnight in growth medium at 10,000 to 40,000 cells/well/90 μL for a 96-well plate or 2,500 to 10,000 cells/well/20 μL for a 384-well plate.

For non-adherent cells

Centrifuge the cells from the culture medium and suspend the cell pellets in culture medium at 50,000-100,000 cells/well/90 µL for a 96-well poly-D lysine plate or 10,000-25,000 cells/well/20 µL for a 384- well poly-D lysine plate. Centrifuge the plate at 800 rpm for 2 minutes with brake off prior to your experiment.
Note Each cell line should be evaluated on an individual basis to determine the optimal cell density.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

ATP Red™ stock solution (500X)

Add 50 µL of DMSO (Component C) into the vial of ATP Red™ (Component A) to make a 500X stock solution.
Note 50 µL of the 500X ATP Red™ stock solution is enough for one 96-well plate. The unused ATP Red™ stock solution can be aliquoted and stored at ≤ -20 °C in smaller aliquots. Protect from light and avoid repeated freeze-thaw cycles.

PREPARATION OF WORKING SOLUTION

ATP Red™ working solution

Add 5 µL of the 500X stock solution (Component A) into 1 mL cell culture medium, and mix well.
Note The ATP Red™ probe is compatible with cell culture media of most cell lines we tested. The staining conditions may be modified according to the particular cell type.

SAMPLE EXPERIMENTAL PROTOCOL

Stain cells

Prepare cells in growth medium.
Add equal volume [100 µL/well (96-well plate) or 25 µL/well (384-well plate)] of ATP Red™ working solution in the cell plate.
Note The optimal concentration of ATP Red™ varies depending on the specific application.
Incubate the cells at 37 °C for 15-30 minutes, protected from light.
Remove working solution from each well. Wash cells with Assay Buffer (Component B) or buffer of your choice twice.
Observe the fluorescence signal in cells using a fluorescence microscope with a Cy3/TRITC filter set.

Images

Figure 1. The fluorescence images of HeLa cells co-stained with ATP Red™ and MitoLite™ Green FM in a 96-well black-wall clear-bottom plate. HeLa cells were stained with ATP Red™ for 15 min and then incubated with 100 nM MitoLite™ Green FM (Cat#22695) for another 30 minutes. Washed twice with assay buffer before imaging. ATP Red™ and MitoLite ATP Red™ signals overlay well. (Far right image) The cells were imaged using a fluorescence microscope with a Cy3/TRITC and FITC filters.

References

View all 50 references: Citation Explorer

Mitochondrial C3a Receptor Activation in Oxidatively Stressed Epithelial Cells Reduces Mitochondrial Respiration and Metabolism.
Authors: Ishii, Masaaki and Beeson, Gyda and Beeson, Craig and Rohrer, Bärbel
Journal: Frontiers in immunology (2021): 628062

Metabolic co-dependence of the oocyte and cumulus cells: essential role in determining oocyte developmental competence.
Authors: Richani, Dulama and Dunning, Kylie R and Thompson, Jeremy G and Gilchrist, Robert B
Journal: Human reproduction update (2021): 27-47

Variability of mitochondrial energy balance across brain regions.
Authors: Cheng, XinPing and Vinokurov, Andrey Y and Zherebtsov, Evgeniy A and Stelmashchuk, Olga A and Angelova, Plamena R and Esteras, Noemi and Abramov, Andrey Y
Journal: Journal of neurochemistry (2020)

The spatio-temporal organization of mitochondrial F1FO ATP synthase in cristae depends on its activity mode.
Authors: Salewskij, Kirill and Rieger, Bettina and Hager, Frances and Arroum, Tasnim and Duwe, Patrick and Villalta, Jimmy and Colgiati, Sara and Richter, Christian P and Psathaki, Olympia E and Enriquez, José A and Dellmann, Timo and Busch, Karin B
Journal: Biochimica et biophysica acta. Bioenergetics (2020): 148091

Intracellular ATP levels influence cell fates in Dictyostelium discoideum differentiation.
Authors: Hiraoka, Haruka and Nakano, Tadashi and Kuwana, Satoshi and Fukuzawa, Masashi and Hirano, Yasuhiro and Ueda, Masahiro and Haraguchi, Tokuko and Hiraoka, Yasushi
Journal: Genes to cells : devoted to molecular & cellular mechanisms (2020): 312-326

Measurement of ATP concentrations in mitochondria of living cells using luminescence and fluorescence approaches.
Authors: Morciano, Giampaolo and Imamura, Hiromi and Patergnani, Simone and Pedriali, Gaia and Giorgi, Carlotta and Pinton, Paolo
Journal: Methods in cell biology (2020): 199-219

Multiparametric imaging reveals that mitochondria-rich intercalated cells in the kidney collecting duct have a very high glycolytic capacity.
Authors: Ghazi, Susan and Bourgeois, Soline and Gomariz, Alvaro and Bugarski, Milica and Haenni, Dominik and Martins, Joana R and Nombela-Arrieta, César and Unwin, Robert J and Wagner, Carsten A and Hall, Andrew M and Craigie, Eilidh
Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology (2020): 8510-8525

Oxidative switch drives mitophagy defects in dopaminergic parkin mutant patient neurons.
Authors: Schwartzentruber, Aurelie and Boschian, Camilla and Lopes, Fernanda Martins and Myszczynska, Monika A and New, Elizabeth J and Beyrath, Julien and Smeitink, Jan and Ferraiuolo, Laura and Mortiboys, Heather
Journal: Scientific reports (2020): 15485

Dynamic regulation of subcellular mitochondrial position for localized metabolite levels.
Authors: Alshaabi, Haya and Heininger, Meara and Cunniff, Brian
Journal: Journal of biochemistry (2020): 109-117

Mitochondria transfer enhances proliferation, migration, and osteogenic differentiation of bone marrow mesenchymal stem cell and promotes bone defect healing.
Authors: Guo, Yusi and Chi, Xiaopei and Wang, Yifan and Heng, Boon Chin and Wei, Yan and Zhang, Xuehui and Zhao, Han and Yin, Ying and Deng, Xuliang
Journal: Stem cell research & therapy (2020): 245