Adenosine triphosphate (ATP) is the primary source of energy that drives cellular metabolism with excess energy stored as glycogen and lipids. When cells are hypoxic, ischemic, structurally damaged, or starved during fasting, the resulting depletion of ATP leads to cellular necrosis, ischemic injury, and postmortem rigidity of muscles known as rigor mortis. Elevated ATP levels is the result of malignant transformation, antibiotic stress, or nutrient and ionic imbalances, ultimately leading to metabolic slowdown, magnesium depletion, and altered protein solubility. Monitoring ATP levels is important in cellular and tissue health evaluation, metabolic toxicity, and advancing diverse scientific research.

ATP Assay Kit provides a simple, reproducible, and sensitive tool of measuring ATP concentration in plasma, serum, cell lysates, urine, saliva, and other biological liquid samples. This assay employs a coupled enzymatic reaction system that combines multiple steps. ATP is hydrolyzed and eventually oxidized with the conversion of the dye into a colored product that is measurable colorimetrically (570 nm) and fluorometrically (Ex/Em = 535/587 nm). The signal measured is directly proportional to the concentration of ATP present in the sample.

• Microplate reader capable of measuring OD 570 nm or Ex/Em = 535/587 nm
• 96-well black plate for fluorometric assay
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Tubes to prepare sample dilutions
• 5 mL tubes
• Multi-channel pipettes to deliver 20 µL to 200 µL volumes
• MilliQ water or another type of double distilled water (ddH₂O)
• Perchloric acid (HClO₄) (PCA)
• Potassium hydroxide (KOH)

1. Add 50 µL of the diluted ATP Standards or samples to the 96-well microtiter plate.
2. Add 50 µL Reaction Mix (+ATPCE) to each standard and one half of the paired sample wells, mix thoroughly.
3. Add 50 µL of Control Mix (-ATPCE) to other half of the paired sample wells. Carefully shake the plate for a few seconds to mix.
4. Incubate the plate for 30 minutes at room temperature with mixing in the dark.
5. Measure the absorbance at 570 nm or fluorescent intensity at Ex/Em 535/587 nm using a plate reader.