Assay ATP in 1 hr in cell/tissue extracts, and biofluids with ATP Assay Kit product, we recommend Luminescent ATP Detection Assay Kit (ab), which can mitochondrial function, and oxidative stress, and also about how to assay. Assay ATP in 30 min in cell samples with Luminescent ATP Detection Assay Kit mitochondrial function, and oxidative stress, and also about how to assay. If I want to simply look at the ATP produced in mitochondria, can I grow . If I just use etiolated plant leaves to perform the luciferine-luciferase assay, is it will Is there anyone who has experience with cellular ATP content measurement?.
detection) Mitochondrial assay (ATP
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We offer numerous convenient solutions to meet your lab's needs. The assay is based on the differential measurement of biomarkers associated with changes in cell membrane integrity and cellular ATP levels relative to vehicle-treated control cells during short exposure periods.
Cell membrane integrity is first assessed by measuring the presence or absence of a distinct protease activity associated with necrosis using a fluorogenic peptide substrate bis-AAF-R to measure "dead cell protease activity". The bis-AAF-R substrate cannot cross the intact membrane of live cells and therefore gives no signal with viable cells. The two sets of data can be combined to produce profiles representative of mitochondrial dysfunction or non-mitochondrial related cytotoxic mechanisms.
Mammalian cells generate ATP by mitochondrial oxidative phosphorylation and non-mitochondrial glycolysis methods. To achieve optimal mitochondrial responsiveness, it may be necessary to refine cell culture conditions.
Replacing glucose-supplemented medium with galactose-containing medium may increase cellular oxygen consumption and augment mitochondrial susceptibility to mitotoxicants. Some cells treated in the presence of glucose may preferentially rely on glycolysis to meet bioenergetic needs and are therefore relatively unresponsive to mitochondrial toxins Glucose ATP.
Cells treated in the presence of galactose must use oxidative phosphorylation to generate ATP and are therefore more responsive to mitochondrial perturbation Galactose ATP.
Oligomycin treatment did not cause changes in membrane integrity in either formulation of medium Galactose Cytotoxicity and Glucose Cytotoxicity. The cells were exposed to oligomycin for 2 hours. No changes in ATP or membrane integrity MI are detected, which indicates that the compound is not a mitochondrial toxin. The reduction in ATP with commensurate MI changes indicate that the compound is not a mitochondrial toxin; instead primary necrosis is taking place.
The reduction in ATP with discordant changes in MI indicate that the compound is a mitochondrial toxin. No parallel processing in glucose-containing medium. Homogeneous assay to quantify total glutathione and glutathione ratios. Sensitive, bioluminescent assay that measures the level of H 2 O 2 in cell cultures. Determine the number of viable cells in culture based on quantitation of ATP present. A single-reagent-addition, homogeneous, fluorescent assay that measures the relative number of dead cells in cell populations.
A single-reagent-addition fluorescent assay that measures the number of live and dead cells in culture wells. High-performance microplate reader for detecting luminescence, fluorescence and absorbance. Our website does not fully support your browser. Your Account Username Account not found. Email address is unverified. Password reset is required. Contact Customer Service Forgot Password?
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BeSO 4 and NaF are prepared as aqueous stock solutions of 0. Vanadate, beryllium, and fluoride salts are highly toxic to tissues and to the environment, and thus require proper handling and disposal. Optimum digitonin amount added to the chamber is determined by measuring oxygen consumption in nonpermeabilized cells using succinate as respiratory substrate, as detailed in Kawamata et al. The entire study should be performed using the same digitonin stock solution.
Emitted light is detected by a photodiode range of sensitivity: A cuvette-based fluorimeter equipped with the capability of stirring and thermoregulation would also be suitable e. The amount of cells required varies from one type to another.
For other cells, such as human fibroblasts, 1—2 million cells are required. Regardless of the cell type, cells are washed once in phosphate-buffered saline and harvested with 0. Next, cells are washed once in buffer A without disturbing the pellet. After the wash, cells are resuspended in 0.
This buffer contains the same base composition as buffer A described above, but includes substrates glutamate and malate as well as inhibitors. See steps below for specific concentrations of substrates and inhibitors to include.
The rationale for using this particular buffer composition is elaborated in Chinopoulos et al. Glutamate and malate as mitochondrial substrates were chosen on the basis that they support mitochondrial substrate-level phosphorylation, and as such, they contribute to greater ATP efflux rates Chinopoulos et al.
C Corrected calibrated trace of panel B , as described in the text. The above equation is available for download as an executable file at http: The rate of ATP appearing in the medium can be calculated by making a linear regression for the ATP values as a function of time, as shown in Fig.
C Reconstructed time recording of oxygen concentration in the medium black trace and oxygen flux gray trace recorded simultaneously with either MgGr signal panel A or safranin O signal panel A. Oxygen concentration black trace of Fig. Using this technique, we have previously addressed ANT-dependent adenine nucleotide exchange in disease conditions Kawamata et al. In the same cells, ATP synthesis measured by a luciferase-based method did not show significant differences between mutant and wild-type ANT1-expressing cells.
Thus, this is a sensitive method to determine ATP production from mitochondria. This may be particularly informative when studying bioenergetic parameters of cancer cell mitochondria, as these are known to exhibit decreased or complete loss of electron flux, which leads to impaired respiration and ATP synthesis Kwong et al.
National Center for Biotechnology Information , U. Author manuscript; available in PMC Nov 2. Author information Copyright and License information Disclaimer. The publisher's final edited version of this article is available at Methods Enzymol.
See other articles in PMC that cite the published article. Abstract We have previously described a fluorometric method to measure ADP—ATP exchange rates in mitochondria of permeabilized cells, in which several enzymes that consume substantial amounts of ATP and other competing reactions interconverting adenine nucleotides are present. Culturing and harvesting of the cells The amount of cells required varies from one type to another. Cells resuspended in 2 ml of buffer C are added to a chamber of an Oroboros Oxygraph-2k.
The presence of cATR and oligomycin in the buffer is only required for the K d determination. MgGr fluorescence signal is recorded upon stepwise additions of 0. This is shown in Fig. Open in a separate window. Continuing in same cells in the chamber , add 0.
Likewise, in a new cell preparation, repeat steps 1—3, but with 11 additions of 0. Convert the MgGr signal of both left and right part of Fig. To do this, plot the steady states of MgGr after each addition of MgCl 2 as seen in the left part of Fig. This is an exponential rise to maximum equation with a three-parameter function, where y 0 , a , and b are coefficients.
The results are shown in Fig. Calibrate the right part of Fig. After calibration of MgGr signals obtained in Fig. If calibration was performed correctly, the left parts of both traces should be almost identical.
Next, from the calibrated right part of Fig. Record MgGr fluorescence for a few minutes and allow the signal to stabilize. In the meantime, harvest cells by trypsinization as elaborated above. Cells are washed once with buffer A and then resuspended in 0. Upon addition of cells to the measuring chamber, wait for 3 min and resume recording. When ADP is added, the fluorescence signal will drop, as shown in Fig.
The value of 0.
Mitochondrial ToxGlo™ Assay
The Mitochondrial ToxGlo™ Assay provides a method for predicting potential Next, ATP is measured by adding an ATP detection reagent, resulting in cell lysis . High-Throughput Assays for Mitochondrial Function ATP assay (B) following treatment with mitochondrial inhibitors (Oligomycin, Rotenone, Antimycin), High-throughput plate assay used to detect mitochondria dysfunction through oxygen. FRET-based ATP biosensor and ATP production assay in a semi- intact cell measurement of the intramitochondrial matrix ATP concentra-.