Adenosine triphosphate (ATP) is primarily synthesized in mitochondria and serves as the "molecular unit of currency" for intracellular energy transfer. ATP plays a fundamental role in cellular energetics, metabolic regulation, cell proliferation, and overall cell health by driving numerous biological processes and chemical synthesis reactions in living cells.

The RayBio^® Live Cell ATP Assay Kit is designed for monitoring intracellular ATP levels in live cells using a cell‑permeable ATP Red™ fluorescent probe. As a boronic acid-ribose covalent structure, the ATP Red™ probe selectively targets and rapidly responds to intracellular ATP concentrations with minimal cross‑reactivity to ADP, AMP, GTP, UTP, and other nucleotides. Real‑time intracellular ATP levels can be measured using a fluorescence microscope or microplate reader equipped with a Cy3/TRITC filter set or the following settings: Ex/Em = 510 nm/590 nm. This kit is suitable for applications such as drug safety screening, mechanistic toxicology, and high‑content analysis.

• ATP Red™ Probe
• Assay Buffer
• ATP Depletion Control
• DMSO

Note: This kit can accommodate up to 40/80 tests if using 24-well plates (for fluorescent microscope measurement) or up to 150/300 tests if using 96-well microplates (for microplate reader measurement). The exact number of tests depends on the seeding volumes and can vary across different cell types and applications.

Note: ATP Depletion Control is a control used to reduce intracellular ATP levels and confirm assay responsiveness.

• Fluorescent plate reader or microscope equipped with a Cy3/TRITC filter set or capable of reading Ex/Em = 510 nm/590 nm
• Pipette tips (barrier tips recommended) and microcentrifuge tubes
• PBS
• ddH₂O
• Centrifuge
• Vortex mixer
• Culture media
• If using a microplate reader, sterile 96-well microplates
• If using a microscope, Sterile round coverslips and 24-well plates
• Mounting medium such as VECTASHIELD^® Mounting Medium
• Coverslip sealant such as clear nail polish

1. Prepare cultured cells as instructed in the manual.
2. Treat cells according to the experimental design. Prepare ATP depletion control and non-stained control wells as needed.
3. Incubate the cells with 1X ATP Red™ Probe working solution.
4. Wash the cells twice with Assay Buffer to remove excess ATP Red™ Probe.
5. Measure fluorescence using a microplate reader or fluorescent microscope equipped with a Cy3/TRITC filter set.

The kit can be stored at 4°C, protected from light, for up to 12 months prior to opening. After reconstitution, aliquot ATP Red and store at -20°C or -80°C, protected from light. Avoid repeated freeze-thaw cycles.