| Description | ATP is the most fundamental energy currency in living organisms, and its concentration directly affects the energy metabolism of various organs. As the most important energy molecule, ATP plays a critical role in diverse physiological and pathological processes. Changes in ATP levels can ATP is the most fundamental energy currency in living organisms, and its concentration directly affects the energy metabolism of various organs. As the most important energy molecule, ATP plays a critical role in diverse physiological and pathological processes. Changes in ATP levels can impact numerous cellular functions. Typically, ATP levels decrease during apoptosis, necrosis, or under certain toxic conditions, while high glucose stimulation can upregulate intracellular ATP levels in some cells. A decrease in ATP levels often indicates impaired or declining mitochondrial function. During apoptosis, the drop in ATP levels usually occurs simultaneously with a decrease in mitochondrial membrane potential. The ATP Assay Kit can be used to detect ATP levels in common solutions, cells, or tissues. This kit is developed based on the principle that firefly luciferase requires ATP to provide energy for catalyzing the production of light from luciferin. When both firefly luciferase and luciferin are in excess, the light produced is proportional to the ATP concentration within a certain range. This allows for highly sensitive detection of ATP concentration in solutions.E1501756Component200TStorageE1501756AATP Detection Reagent25 mL-20℃. Store in the dark.E1501756BATP Standard Solution100 µL-20℃. Store in the dark.E1501756CATP Assay Lysis Buffer100 mL-20℃. Store in the dark.Product Advantages1. High Sensitivity: Provides excellent detection results within the range of 0.1 nM to 100 µM.2. High Stability: ATP measurement results from prepared samples decrease by no more than 10% within 30 minutes.3. Good Compatibility of Prepared Samples: Cell or tissue samples lysed using the ATP Assay Lysis Buffer provided in this kit can not only be used for ATP detection but also for protein concentration assays, SDS-PAGE, or Western blotting for some commonly soluble proteins.4. Convenient and Fast: Typically, 10-20 samples can be assayed within 30-60 minutes.5. Simple Sample Preparation: Samples do not require perchloric acid or trichloroacetic acid (TCA) extraction. The specialized lysis buffer provided allows samples to be used for ATP detection after simple lysis.Experimental Procedure1. Sample PreparationNote: Sample lysis should be performed at 4°C or on ice.1.1 For Adherent CellsRemove the culture medium. Add Lysis Buffer according to the proportion of 200 µL per well of a 6-well plate (i.e., 1/10 of the 2 mL culture medium volume) to lyse the cells. For complete lysis, pipette up and down repeatedly or shake the plate to ensure the lysis buffer fully contacts and lyses the cells. Cells typically lyse immediately upon contact with the buffer. Centrifuge the lysate at 10,000 rpm, 4°C for 5 minutes. Collect the supernatant for subsequent assay.1.2 For Suspension CellsCentrifuge to pellet the cells, discard the supernatant, and gently resuspend the pellet. Add Lysis Buffer according to the proportion of 200 µL per the cell amount from one well of a 6-well plate. For complete lysis, tap the tube bottom or vortex appropriately to ensure the lysis buffer fully contacts and lyses the cells. Cells typically lyse immediately. Centrifuge the lysate at 10,000 rpm, 4°C for 5 minutes. Collect the supernatant for subsequent assay.1.3 For Tissue SamplesAdd Lysis Buffer in a ratio of approximately 100-200 µL per 20 mg of tissue. Homogenize using a glass homogenizer or other homogenization equipment. Thorough homogenization ensures complete tissue lysis. Centrifuge the lysate at 10,000 rpm, 4°C for 5 minutes. Collect the supernatant for subsequent assay.2. Standard Curve PreparationThaw the required reagents on ice. Dilute the ATP Standard Solution with ATP Assay Lysis Buffer to create appropriate concentration gradients. The specific concentrations should be determined based on the expected ATP concentration in the samples. For initial detection, concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 µM can be tested. In subsequent experiments, adjust the standard concentration range appropriately based on sample ATP levels.TubeLysis Buffer Volume (µL)ATP Standard Solution VolumeFinal Concentration (µM)A982 µL from stock (0.5 mM)10B7030 µL from Tube A3C9010 µL from Tube A1D9010 µL from Tube B0.3E9010 µL from Tube C0.1F9010 µL from Tube D0.03G9010 µL from Tube E0.013. ATP Concentration Measurement3.1 Add 100 µL of ATP Detection Reagent to each assay well. Incubate at room temperature for 3-5 minutes.3.2 Add 10 µL of sample or the diluted ATP standard solution to the assay well.3.3 Measure the Relative Light Unit (RLU) value using a luminometer.Note: The sample volume can be adjusted within the range of 10-100 µL. If the ATP concentration in the sample is low, 100 µL can be added. If the ATP concentration is high, a smaller volume can be used, but the same volume must be used for the standard curve samples. If the ATP concentration is exceptionally high, dilute the sample with ATP Assay Lysis Buffer before measurement.Precautions1. The Detection Reagent contains luciferase. Repeated freeze-thaw cycles will lead to gradual inactivation. For optimal performance, consider aliquoting after the first thaw, ensuring the aliquot containers are free from ATP contamination.2. Luciferase activity is temperature-sensitive. Before the reaction, equilibrate both cells and the ATP Detection Reagent to room temperature for measurement. Do not store at room temperature for extended periods.3. ATP, especially in lysed samples, is unstable at room temperature. Perform operations at 4°C or on ice.4. Use white or black 96-well or 384-well plates suitable for cell culture for detection. Using standard transparent plates may cause interference between adjacent wells.5. The provided ATP Assay Lysis Buffer effectively lyses and releases ATP from common cultured cells and tissues. For special tissues or samples where detected ATP levels are significantly lower than expected, boil a portion of the lysate for 2 minutes before centrifugation to fully release ATP. Boiling will denature proteins, which will precipitate during subsequent centrifugation; therefore, boiled samples cannot be used for protein concentration assays, SDS-PAGE, or Western blotting. Use the remaining portion of the sample for protein assays, SDS-PAGE, and Western blotting.6. 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