Description
Intracellular Cuprous Ion (Cu+) Fluorometric Assay Kit (MAES0539)
The Intracellular Cuprous Ion (Cu+) Fluorometric Assay Kit provides a rapid and sensitive method for detecting free cuprous ion (Cu+) levels in living cultured cells. Cu+ is an essential trace element involved in energy metabolism and antioxidant defense, but excess free intracellular Cu+ binds to lipoylated proteins, triggering their oligomerization and loss of function, proteotoxic stress and ultimately cuproptosis. Monitoring the dynamic homeostasis of intracellular Cu+ is therefore critical for research on copper metabolism, cuproptosis, oxidative stress and drug response.
In this assay, a specific fluorescent cuprous probe with weak intrinsic fluorescence is loaded into suspension or adherent cells. Upon specific coordination with Cu+, the probe undergoes a conformational change that markedly increases its red fluorescence. Signal is read on a flow cytometer (PE-Texas Red channel) or a fluorescence microscope (Ex/Em = 480–500 nm/610–650 nm), using a simple probe-staining step of 5–20 minutes at 25°C in the dark.
| Product Name: | Intracellular Cuprous Ion (Cu+) Fluorometric Assay Kit |
| SKU: | MAES0539 |
| Detection Method: | Fluorometric Method (Fluorescent Probe) |
| Sample Type: | Cultured Cells (Suspension and Adherent Cells) |
| Detection Instrument: | Flow Cytometer (PE-Texas Red Channel), Fluorescence Microscope (Ex/Em = 480–500 nm/610–650 nm; PE Filter Set) |
| Research Area: | Cell Biology, Copper Metabolism, Cuproptosis |
| Storage: | Store at room temperature, protected from light, for 12 months. |
| Sample Volume: | 0.2 mL cell suspension per well (suspension cells); 0.5 mL PBS per well (adherent cells) |
| Assay Time: | ~5–20 min staining |
| Target: | Free Intracellular Cuprous Ion (Cu+) |
| Applications: | Flow Cytometry, Fluorescence Microscopy (Confocal or Conventional) |
| Excitation/Emission: | Ex: 480–500 nm / Em: 610–650 nm (Red Fluorescence) |
| Staining Conditions: | 25°C in the dark for 5–20 min (optimize per cell line, starting at 5 min) |
This kit utilizes a specific fluorescent cuprous probe to detect intracellular Cu+ levels. The probe itself exhibits weak intrinsic fluorescence. Upon specific coordination with Cu+, the molecule undergoes a conformational change, resulting in a marked increase in fluorescence intensity. The fluorescence signal, detected by flow cytometry or fluorescence microscopy, reflects the level of free Cu+ within the cells.
| Step | Procedure |
| 1 | Cell Preparation: Suspension cells – collect by centrifugation, resuspend in PBS at ≥1×105 cells/mL and add 0.2 mL per well of a 96-well plate. Adherent cells – seed at ≥1×104 cells/mL in a 24-well plate, culture overnight at 37°C with 5% CO2, wash twice with PBS and add 0.5 mL PBS per well. Include a blank control (no probe) and at least three replicate wells per group. |
| 2 | Probe Loading: Add probe solution to experimental wells (1 µL per well for suspension cells; 2.5 µL per well for adherent cells) and an equal volume of culture medium to blank control wells. |
| 3 | Staining: Incubate at 25°C in the dark for 5–20 min. Start with 5 min and optimize the staining time for each cell line. |
| 4 | Flow Cytometry: For adherent cells, remove the supernatant, digest with 0.2 mL trypsin and stop with 0.4 mL complete medium. Acquire at least 1×104 cells per sample in the PE-Texas Red channel. |
| 5 | Fluorescence Microscopy: Remove the medium, wash cells twice with PBS, add fresh PBS and image (confocal Ex/Em = 480–500 nm/610–650 nm, or a PE filter set on a conventional microscope). |
