ADP, ATP Ratio Bioluminescence Assay Kit, ApoSENSOR

Details for Product No. ABIN411720
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Reactivity
Mammalian
Application
Bioluminescence Assay (BioLumA)
Pubmed 21 references available
Quantity 200 tests
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Purpose The changes in ADP/ATP ratio have been used to differentiate the different modes of cell death and viability. Increased levels of ATP and decreased levels of ADP have been recognized in proliferating cells. In contrast, decreased levels of ATP and increased levels of ADP are recognized in apoptotic cells. The decrease in ATP and increase in ADP are much more pronounced in necrosis than apoptosis. The ApoSENSORTM ADP/ATP Ratio Assay kit utilizes bioluminescent detection of the ADP and ATP levels for a rapid screening of apoptosis, necrosis, growth arrest, and cell proliferation simultaneously in mammalian cells. The assay utilizes the enzyme luciferase to catalyze the formation of light from ATP and luciferin, and the light can be measured using a luminometer or Beta Counter. ADP level is measured by its conversion to ATP that is subsequently detected using the same reaction. The assay can be fully automatic for high throughput and is highly sensitive (detects 100 mammalian cells/well).
Brand ApoSENSOR™
Sample Type Cell Lysate, Tissue Lysate, Serum, Plasma, Urine, Cell Culture Supernatant, Soil, Waste Water
Detection Method Fluorometric
Specificity The ApoSENSOR TM ADP/ATP Ratio Assay kit utilizes bioluminescent detection of the ADP and ATP levels for a rapid screening of apoptosis, necrosis, growth arrest, and cell proliferation simultaneously in mammalian cells. The assay utilizes the enzyme luciferase to catalyze the formation of light from ATP and luciferin, and the light can be measured using a luminometer or Beta Counter. ADP level is measured by its conversion to ATP that is subsequently detected using the same reaction. The assay can be fully automatic for high throughput and is highly sensitive (detects 100 mammalian cells/well).
Characteristics ApoSENSORTM ADP,ATP Ratio Assay Kit: Bioluminescent Detection of ADP & ATP Levels for Rapid Screening of Apoptosis, Necrosis, Growth Arrest and Cell Proliferation Simultaneously in Mammalian Cells. Highly Sensitive & High-Throughput.
Components Nucleotide Releasing Buffer
ATP Monitoring Enzyme
ADP Converting Enzyme
Enzyme Reconstitution Buffer
Background The changes in ADP/ATP ratio have been used to differentiate the different modes of cell death and viability. Increased levels of ATP and decreased levels of ADP have been recognized in proliferating cells. In contrast, decreased levels of ATP and increased levels of ADP are recognized in apoptotic cells. The decrease in ATP and increase in ADP are much more pronounced in necrosis than apoptosis.
Application Notes Bioluminescent detection of the ADP and ATP levels for a rapid screening of apoptosis, necrosis, growth arrest, and cell proliferation simultaneously in mammalian cells.
Comment

Luminometer or Beta Counter.
Simple one-step procedure, takes only 30 minutes
Fast and convenient
The ADP/ATP ratio assay offers highly consistent results and with excellent correlation to other apoptosis markers (e.g. TUNEL-based assays and caspase assays). In addition the assay can be fully automatic for high throughput (10 seconds/sample) and is highly sensitive (detects 10-100 cells/well).

Assay Time 0.5 h
Protocol A. Reagent Reconstitution and General Consideration: Reconstitute ATP Monitoring Enzyme and ADP Converting Enzyme each with 220 µL of the Enzyme Reconstitution Buffer. Mix gently by inversion (Note: The reconstituted Enzymes will be milky, cloudy solution, not clear solution). Aliquot enough enzymes (1 µL per assay) for the number of assays to be performed in each experiment and freeze immediately at -70 °C for future use. The reconstituted enzymes are stable for up to 3 months at -70 °C after reconstitution. For more accurate handling, the enzyme can be dilute 10 fold with Nucleatide Releasing Buffer just before use, then use 10 µL of the enzymes each for each assay. The ApoSENSOR TM kit is significantly more sensitive than other methods used for cell viability assays. The method can detect as few as 10 cells, but as a general guide, we recommend using 1 x 10 4 cells per assay. Avoid contamination with ATP from exogeneous biological sources, such as bacteria or fingerprints. Ensure that the Nucleotide Releasing Buffer is at room temperature before use. The optimal temperature is 22 °C. Keep other enzymes on ice during the assay and protect from light as much as possible. The assay can be performed using either a single tube or a white walled 96-well luminometer plate (100 µL/well culture volume is recommended).
1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction.
2. For suspension cells, transfer 10 µL of the cultured cells (10 3 - 10 4 ) into luminometer plate. Add 100 µL of the Nucleotide Releasing Buffer. For adherent cells, remove culture medium and treat cells (10 3 - 10 4 ) with 100 µL of Nucleotide Releasing Buffer for 5 minutes at room temperature with gentle shaking.
3. To measure the ATP levels in the cells, add 1 µL of the ATP Monitoring Enzyme into the cell lysate. Read the sample in 1 minute in a luminometer (Data A).
4. To measure ADP levels in the cells, read the samples (from step 3) in 10 minutes (Data B), then add 1 µL of ADP Converting Enzyme. Read the samples again in 1 minute in a luminometer (Data C). Note: The reconstituted Enzymes will be milky, cloudy solution, not clear solution). Aliquot enough enzymes (1 µL per assay) for the number of assays to be performed in each experiment and freeze immediately at -70 °C for future use. The reconstituted enzymes are stable for up to 3 months at -70 °C after reconstitution. For more accurate handling, the enzyme can be dilute 10 fold with Nucleatide Releasing Buffer just before use, then use 10 µL of the enzymes each for each assay.
The ApoSENSOR™kit is significantly more sensitive than other methods used for cell viability assays. The method can detect as few as 10 cells, but as a general guide, we recommend using 1 x 10 4 cells per assay. Avoid contamination with ATP from exogeneous biological sources, such as bacteria or fingerprints.
Ensure that the Nucleotide Releasing Buffer is at room temperature before use. The optimal temperature is 22 °C. Keep other enzymes on ice during the assay and protect from light as much as possible.
The assay can be performed using either a single tube or a white walled 96-well luminometer plate (100 µL/well culture volume is recommended). Assay Protocol:
Restrictions For Research Use only
Storage -80 °C
Expiry Date 12 months
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Kawano, Ersoy, Li et al.: "Thioesterase superfamily member 2 (Them2) and phosphatidylcholine transfer protein (PC-TP) interact to promote fatty acid oxidation and control glucose utilization." in: Molecular and cellular biology, Vol. 34, Issue 13, pp. 2396-408, 2014 (PubMed).

Tsai, Greco, Cristea: "Sirtuin 7 plays a role in ribosome biogenesis and protein synthesis." in: Molecular & cellular proteomics : MCP, Vol. 13, Issue 1, pp. 73-83, 2014 (PubMed).

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Geng, Fu, Ji et al.: "Synergetic neuroprotection of normobaric oxygenation and ethanol in ischemic stroke through improved oxidative mechanism." in: Stroke; a journal of cerebral circulation, Vol. 44, Issue 5, pp. 1418-25, 2013 (PubMed).

Timofeeva, Tarasova, Zhang et al.: "STAT3 suppresses transcription of proapoptotic genes in cancer cells with the involvement of its N-terminal domain." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 110, Issue 4, pp. 1267-72, 2013 (PubMed).

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Cerchietti, Polo, Da Silva et al.: "Sequential transcription factor targeting for diffuse large B-cell lymphomas." in: Cancer research, Vol. 68, Issue 9, pp. 3361-9, 2008 (PubMed).

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Mimura, Joyce: "Replication competence and senescence in central and peripheral human corneal endothelium." in: Investigative ophthalmology & visual science, Vol. 47, Issue 4, pp. 1387-96, 2006 (PubMed).

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Catalog No. ABIN411720
517.00 $
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200 tests
517.00 $

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