Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins.

BACKGROUND: The combination of fluorescence resonance energy transfer (FRET) and flow cytometry offers a statistically firm approach to study protein associations. Fusing green fluorescent protein (GFP) to a studied protein usually does not disturb the normal function of a protein, but quantitation of FRET efficiency calculated between GFP derivatives poses a problem in flow cytometry. METHODS: We generated chimeras in which cyan fluorescent protein (CFP) was separated by amino acid linkers of different sizes from yellow fluorescent protein (YFP) and used them to calibrate the cell-by-cell flow cytometric FRET measurements carried out on two different dual-laser flow cytometers. Then, CFP-Kip1 was coexpressed in yeast cells with YFP and cyclin-dependent kinase-2 (Cdk2) and served as a positive control for FRET measurements, and CFP-Kip1 coexpressed with a random peptide fused to YFP was the negative control. RESULTS: We measured donor, direct, and sensitized acceptor fluorescence intensities and developed a novel way to calculate a factor (alpha) that characterized the fluorescence intensity of acceptor molecules relative to the same number of excited donor molecules, which is essential for quantifying FRET efficiency. This was achieved by calculating FRET efficiency in two different ways and minimizing the squared difference between the two results by changing alpha. Our method reliably detected the association of Cdk2 with its inhibitor, Kip1, whereas the nonspecific FRET efficiency between Cdk2 and a random peptide was negligible. We identified and sorted subpopulations of yeast cells showing interaction between the studied proteins. CONCLUSIONS: We have described a straightforward novel calibration method to accurately quantitate FRET efficiency between GFP derivatives in flow cytometry.

Neurochemical diagnosis of Alzheimer's dementia by CSF Abeta42, Abeta42/Abeta40 ratio and total tau.

Cerebrospinal fluid (CSF) concentrations of amyloid beta peptides ending at positions 42 and 40 (Abeta42 and Abeta40, respectively), and total tau (tTau) protein were measured by ELISA in order to compare their accuracy in discriminating patients with Alzheimer's disease (AD, n = 22), non-Alzheimer dementia (nAD, n = 11) and control subjects (CON, n=35). As compared to the other groups, the concentrations of Abeta42 and tTau were decreased (P<0.001) and increased (P<0.001) in AD, respectively, while Abeta40 did not differ significantly among the groups. Receiver operating characteristic (ROC) analysis was performed to define cut-off values for maximized sensitivity and specificity. For all groups compared the Abeta peptide ratio 42/40 classified more patients correctly, as compared to the concentration of Abeta42 alone: AD versus controls, 94 and 86.7%; AD versus nAD, 90 and 85% and AD versus nAD plus controls, 90.8 and 87%, respectively. The percentage of correctly classified patients was further improved when the Abeta ratio was combined with the analysis of the tTau concentration. Presence of the apolipoprotein E 4 allele, age or degree of mental disability did not significantly influence the parameters studied.