(229al) Cytometric Measurement of Caspase-3 Activation Using Forster Resonance Energy Transfer (FRET) Between Peptide-Linked Bioprobes

Fluorescence lifetime measurements with flow cytometry provide high throughput and quantitative information about molecules within cells based on fluorescence intensity decay times. This time-resolved cytometry method is not commercially available but can be introduced onto any cytometry system, as our laboratory has done. Fluorescence lifetime measurements are helpful for a range of applications including the measurement of Forster Resonance Energy Transfer (FRET). In this contribution we screen large cell populations with our cytometry instrument in order to identify the induction of apoptosis (programmed cell death) by quantifying the activity of caspase-3 with a FRET assay.

Methods

A FRET based bioprobe is engineered with green fluorescent protein as well as a caspase-3 recognition sequence. Our modified GFP, which is the FRET donor is connected to an exogenous fluorophore (based on spectral properties: AlexaFluor® Dye, Thermo Fisher Scientific Inc.), which acts as the acceptor in the FRET pair. A Caspase-3 recognition peptide linker site connects the FRET pair such that during the induction of apoptosis, caspase-3 is present and available for cleavage of the peptide. Our measurements include excitation and emission spectra before and after caspase-3 activation with a fluorimeter. Additionally we detect fluorescence lifetime changes using our flow cytometer before and after caspase-3 activation.

Results

Our analysis of the emission spectra confirm the presence and loss of FRET prior and post caspase-3 treatment respectively. We calculated the emission ratios representing maximum of dye emission to maximum of modified GFP emission. Furthermore the observed change in fluorescence lifetimes measured before and after caspase-3 activation help us discriminate the loss of FRET accurately.

Conclusions

Fluorescence lifetime measurements on a flow cytometer is a powerful method to provide a quantifiable way to study apoptosis in mammalian cells grown in a monolayer culture. This assay permits us to determine quantities of caspase-3 through the measurement of FRET as well as loss of FRET.