AAT Bioquest

What are the principles of 5-CFDA-AM assay?

Posted June 21, 2023


5-CFDA-AM assay is based on the principle of measuring cellular esterase activity, which is an indicator of cell viability

 5-CFDA-AM or 5-Carboxyfluorescein Diacetate, Acetoxymethyl Ester) is a non-fluorescent cell-permeable dye that can enter live cells. Once inside the cell, cellular esterases cleave the AM (acetoxymethyl) groups from 5-CFDA-AM, resulting in the formation of 5-carboxyfluorescein (5-CF). 

5-CF is a highly fluorescent compound that remains trapped within the cells, indicating their viability as only an intact membrane can maintain the cytoplasmic environment necessary for supporting esterase activity

The fluorescence emitted by 5-CF can be quantified using fluorescence microscopy or flow cytometry, providing a measure of live cell population.

Additional resources

A comparison of TO-PRO-1 iodide and 5-CFDA-AM staining methods for assessing viability of planktonic algae with epifluorescence microscopy

Cell Viability Assays

CFDA [5-(and 6)-Carboxyfluorescein diacetate] *Mixed isomers*