Cell Counting Kit-F(CCK-F) is utilized for the fluorometric determination of living cell numbers. The amount of the fluorescent dye, calcein, hydrolyzed by esterases in cells is directly proportional to the number of viable cells in culture media (Fig. 1). Since esterases and phenol red in the culture medium interfere with the fluorescence measurement, replacing the cell culture medium with PBS is necessary prior to adding the Calcein-AM assay solution. The excitation and the emission wavelengths of calcein are 485 nm and 535 nm, respectively (Fig. 3). An incubation of 10 to 30 minutes gives sufficient fluorecence intensity for the cell viability determination.
Fig. 1 Cell viability detection mechanism with CCK-F
Fig. 2 Typical calibration curve using CCK-F
Cell line: HL60
Culture medium: RPMI1640, 10% FCS, L-glutamine
Incubation: 37 °C, 5% CO2, 30 min.
Detection: 485 nm excitation, 535 nm emission
Fig. 3 Fluorescence spectrum of Calcein/ PBS solution, pH 7.4
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