Effect of fixation procedures on the fluorescence lifetimes of Aequorea victoria derived fluorescent proteins.

Fluorescence lifetime imaging microscopy can be used to study protein-protein interactions by Förster Resonance Energy Transfer or to perform lifetime-based multiplexing. Fixation of samples with cells producing fluorescent fusion proteins is commonly used for preservation of samples and for staining with membrane impermeable reagents such as antibodies. However, the effect of fixation methods and mounting media on fluorescence lifetime is poorly documented so far. Here, we demonstrate that fixation by formaldehyde or methanol itself does not affect the lifetime of fluorescent proteins produced in cells but that several widely used mounting media decrease the fluorescence lifetime by up to 20%. It is shown that fixed cells producing Aequorea victoria derived fluorescent proteins mounted in Tris buffer have fluorescence lifetimes indistinguishable from values measured in living cells. Tris buffer also allows accurate Förster Resonance Energy Transfer quantification in fixed cells, as shown with an mTurquoise2-SYFP2 fusion protein. Moreover, identical lifetime contrasts are measured in living and fixed cells mounted in Tris buffer after introducing a single plasmid expressing two lifetime variants of cyan fluorescent proteins, each targeted to different locations in the cell. Our findings will aid the preparation of fixed cells producing fluorescent proteins for reliable measurement of fluorescence lifetimes for Förster Resonance Energy Transfer determination, lifetime based multiplexing and for instrument calibration for standardization purposes.

Abundant expression of homeobox genes in mouse embryonal carcinoma cells correlates with chemically induced differentiation.

Mammalian homeobox-containing genes might play a role in embryonal pattern formation. In favor of this view is the recently reported expression of such genes during mouse embryogenesis [Manley, J. L. & Levine, M. S. (1985) Cell 43, 1-2]. The embryo-derived stem cells and in particular the pluripotent embryonal carcinoma (EC) cell lines are generally considered as a valid model of early mouse development. Homeobox-containing genes were shown to be expressed in differentiating EC cells. We have analyzed the expression of several of these genes in three EC cell lines triggered to differentiate by alternative treatments in the presence or in the absence of retinoic acid. In both types of conditions, C17S1 (clone 1003) and PCC7.S Aza R1 EC cells were induced to differentiate into mainly neurones, and PSA-1 EC cells were induced to differentiate into a large spectrum of tissue derivatives. Induction to high levels of expression of several homeobox-containing genes during differentiation occurs only in the presence of retinoic acid. Nonchemical treatment triggering differentiation does not lead to detectable expression of these genes. Accumulation to high amounts of homeobox-containing gene transcripts in these experiments seems to correlate with retinoic acid-induced EC cell differentiation rather than with EC cell differentiation as such.