Cell Ferrous Iron (Fe²⁺) Assay Kit (Fluorometric) 

Iron is one of the essential metal elements in organism and plays a crucial role in various physiological functions. Ferrous ion is a key component of heme and hemoglobin and plays an important role in many biochemical reactions. In recent years, with the introduction of the concept of ferroptosis, it has been discovered that the absorption, transportation, storage and utilization of iron ions and their excessive accumulation in cells have a significant relationship with aging and a range of diseases.

MCE Cell Ferrous Iron (Fe²⁺) Assay Kit (Fluorometric) utilizes fluorescence detection technology to analyze the levels of ferrous ions (Fe²⁺) in cells. The detection principle is as follows: the fluorescence probe enters the cell and specifically binds to intracellular Fe²⁺, forming an irreversible orange-red fluorescent product (Ex/Em = 543/580 nm). The Fe²⁺ content in cells is determined by measuring the fluorescence signal. For 96-well plate, the 100 T can analyze at least 100 samples.

-20℃, 6 months

Keep away from light

1. Ensure the fluorescent probe is fully dissolved and mixed thoroughly before use. It is recommended to aliquot the solution appropriately to avoid repeated freeze-thaw cycles.

2. This product is suitable for live cell detection; it does not perform effectively in dead cells.

3. It is recommended to set up control groups to observe changes in fluorescence intensity. For the negative control, add an iron chelator (e.g., 2,2’-Bipyridine, Bpy, MCE Cat. No.: HY-D0020); for the positive control, add ammonium ferrous sulfate.

4. Fluorescent dyes are subject to quenching issues. Please ensure to protect them from light to slow down fluorescence quenching.

5. This product is for R&D use only, not for drug, household, or other uses.

6. For your safety and health, please wear a lab coat and disposable gloves to operate.

Since staining conditions vary depending on cell type and cell concentration, it is recommended to perform pre-experiments to determine the optimal concentrations of fluorescence probe. For routine cell staining, the working concentration of the fluorescent probe can be adjusted between 1 - 5 μM.

The following procedure uses a working concentration of 1 μM for the fluorescent probe as an example (the optimal concentration can also be determined through preliminary experiments).

Note: To ensure sufficient signal, it is recommended to use the lowest possible concentration of the fluorescent probe.

1. Reagent Preparation: Remove the fluorescent probe and staining buffer solution, equilibrate at room temperature for 30 min.

Note: For first-time use, it is recommended to aliquot the fluorescent probe appropriately to avoid repeated freeze-thaw cycles.

2. Preparation of Staining Working Solution: Take an appropriate amount of staining buffer and dilute the fluorescent probe 2,000 times, mixing thoroughly to prepare the staining working solution with a final concentration of 1 μM.

Note: a. Calculate the required volume of staining working solution based on the number of samples and the type of plate. For example, for a 96-well plate, 100 μL of staining working solution is needed per well.

b. The staining working solution is recommended to be prepared fresh and used on the same day. Protect the solution from light after preparation.

For Adherent Cells

1. Seed adherent cells into cell culture plates, microplates, or prepare cell coverslips. Incubate overnight in a cell culture incubator.

Note: Suspended cells can also be prepared as cell coverslips and stained following the steps for adherent cells.

2. Discard the supernatant and wash the cells 2 - 3 times with serum-free medium or PBS.

3. Treat the cells with the desired experimental drugs, and wash the cells 2 - 3 times with staining buffer solution.

4. Add an adequate amount of staining working solution, ensuring that the monolayer cells are fully covered.

5. Incubate at 37°C in a cell culture incubator for 20 - 60 min.

Note: The generated fluorescent product is prone to quenching; it is recommended to complete the detection within 2 h after incubation.

For Suspended Cells (Example using a 96-well plate)

1. Centrifuge at 1,000 rpm for 3 min, discard the supernatant, collect the cells, and wash the cells 2 - 3 times with serum-free medium or PBS.

2. After treating the cells with the desired experimental drugs, centrifuge at 1,000 rpm for 3 min, discard the supernatant, collect the cells, and wash the cells 2 - 3 times with staining buffer solution.

Note: Recommended cell quantity is 1 × 10⁴ - 1 × 10⁵.

3. Resuspend the cell pellet in 100 μL of staining working solution and incubate at 37°C in a cell culture incubator for 20 - 60 min.

Note: The generated fluorescent product is prone to quenching; it is recommended to complete the detection within 2 h after incubation.

For Adherent Cells: After staining incubation, there is no need to wash the cells. Observe the cells directly under a fluorescence microscope (Ex/Em = 543/580 nm).

For Suspended Cells: After staining incubation, there is no need to wash the cells. Perform detection directly using a fluorescence microplate reader or flow cytometer (Ex/Em = 543/580 nm).

Note: After staining incubation, it is not necessary to replace the culture medium or wash the cells, as replacing the medium may cause the probe to leak into the extracellular space, affecting the detection results.

1. Ensure the fluorescent probe is fully dissolved and mixed thoroughly before use. It is recommended to aliquot the solution appropriately to avoid repeated freeze-thaw cycles.

2. This product is suitable for live cell detection; it does not perform effectively in dead cells.

3. It is recommended to set up control groups to observe changes in fluorescence intensity. For the negative control, add an iron chelator (e.g., 2,2’-Bipyridine, Bpy, MCE Cat. No.: HY-D0020); for the positive control, add ammonium ferrous sulfate.

4. Fluorescent dyes are subject to quenching issues. Please ensure to protect them from light to slow down fluorescence quenching.

5. This product is for R&D use only, not for drug, household, or other uses.

6. For your safety and health, please wear a lab coat and disposable gloves to operate.