FDA [Fluorescein diacetate] *CAS 596-09-8*

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Chemical structure for FDA [Fluorescein diacetate] *CAS 596-09-8*
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Ex/Em (nm)494/521
CAS #596-09-8
Storage Freeze (<-15 °C)
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Category Cell Biology
Labeling Cells
Related Viability and Proliferation
Cell Viability
Secondary Reagents
FDA is a non-fluorescent hydrophobic fluorescein derivative that can pass through the cell membrane whereupon intracellular esterases hydrolyze the diacetate group producing the highly fluorescent product fluorescein. The fluorescein molecules accumulate in cells that possess intact membranes so the green fluorescence can be used as a marker of cell viability. Cells that do not possess an intact cell membrane or an active metabolism may not accumulate the fluorescent product and therefore do not exhibit green fluorescence. FDA may be used in combination with PI staining as the non-viable cells take up the PI and stain dead cells red whereas viable cells do not take up the PI and should only stain green. This 2-color separation of non-viable and viable cells may provide a more accurate quantitation of cell viability than single color analysis.

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of FDA [Fluorescein diacetate] *CAS 596-09-8* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

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Table 2. Enter any two values (mass, volume, concentration) to calculate the third.

Mass Molecular weight Volume Concentration Moles
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This protocol only provides a guideline, and should be modified according to your specific needs.

1. Prepare 2-10 mM DMSO stock solution

For #22014 add 45 uL DMSO into a 50 µg vial to make 2  mM stock solution (1 mg/ml is equivalent to 1.8 mM);

For #22015 add 36 uL DMSO into a 50 µg vial to make 10 mM stock solution (1 mg/ml is equivalent to 1.46 mM);

For #22016 add 153 uL ml DMSO to make 10 mM stock solution (1 mg/ml is equivalent to 1.53 mM);

For #22017 add 215 uL ml DMSO to make 10 mM stock solution (1 mg/ml is equivalent to 2.15 mM);

For #22020 dissolve 4.2 mg in 1 ml DMSO to make 10 mM stock solution (1 mg/ml is equivalent to ~ 2.4 mM);


Note: The stock solution should be used promptly; any remaining solution should be aliquoted and frozen at < -20 oC. Avoid repeated freeze-thaw cycles, and protect from light.


2. Prepare dye working solution

Prepare a 1 to 20 ┬ÁM dye working solution right before use by diluting the DMSO stock solution from Step 1 with Hanks and 20 mM Hepes buffer (HHBS) or the buffer of your choice, pH 7. Mix them well by vortexing.


3. Analyze cells with a flow cytometer or a fluorescence microscope:

3.1    Treat cells with test compounds for a desired period of time.


3.2    Centrifuge the cells to get 2-10 x105 cells per tube.


3.3     Resuspend cells in 500 µL of the dye working solution (from Step 2).


3.4     Incubate cells with a dye solution at room temperature or 37 °C for 15 to 30 min, protected from light.


3.5     Remove the dye working solution from the cells, wash the cells with HHBS or buffer of your choice. Resuspend cells in 500 µL of pre-warmed HHBS or medium to get 2-10 x 105 cells per tube.


3.6     Monitor the fluorescence change at Ex/Em = 490/520 nm with a flow cytometer (FL1 channel) or a fluorescence microscope.


Note: For bacterial cells staining: Staining is most efficient when stock solution is diluted 1:800 in nutrient broth preconditioned by overnight growth of the test bacteria, but fresh nutrient broth or PBS may also be used. Bacterial suspensions should be diluted with PBS to 105 - 107 organisms per ml. Bacteria may be stained by applying one ml of solution to .45 µm filter (25mm) and vacuum filtering to remove solution, then adding 1ml of dye solution and incubating 5 - 10 minutes at room temperature.

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