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admin | Category: Virtual Phone Numbers | 26.04.2013
Cell proliferation can now be measured flow cytometrically by the use of numerous probes including, Carboxyfluorescein Diacetate, Succinimidyl Ester or CFSE, PHK26, and now Violet Cell Trace, enabling researchers to easily monitor the proliferation rate of cells transfected with GFP, the original CFSE being in the same channel as GFP. CFSE proliferation studies can be combined with live cell cycle analysis throughout a time course study of several days. This new probe, Violet Cell Trace is easily loaded into cell lines such as Jurkat T-cells which can be monitored for proliferation for up to 10 days and be easily imaged by confocal microscopy, although at high concentrations this probe can bleed into the GFP channel.
Human PBMNC lymphocytes were pre-labeled with 1 mM Violet Cell Trace for 20 mins at 37C.Lymphocytes were then PHA activated for 5 days. If violet laser diodes are not available, a Red HeNe diode can be used to measure cell proliferation by the use of Cell Trace Far Red DDAO-SE.


Cell are labelled with the dye CFDA,SE which spontaneously and irreversibly couples to both intracellular and cell surface proteins. Jurkat cells loaded with 5, 2 or 1mM Cell Trace Violet were compared over a 10 day incubation period with fluorescence intensities compared for the 3 concentrations, see figures for 1, 2, 5mM. The violet excitation and blue emission of CellTrace Violet ensures flexibility in Moreexperimental design as it can be used with a wide range of blue-excitable fluorophores, including GFP-expressing cells.
When cells divide CFDA,SE labelling is distributed equally between the two daughter cells, which therefore have half the fluorescence of the parents. Jurkat T-cells were studied for cell proliferation and live cell cycle analysis over a 5 day period, the cell cycle analysis shows that the number of proliferating cells changes over the 5 day period, see figure.


As a result, each successive generation in a population is marked by a halving of the cellular fluorescence intensity which is readily followed by flow cytometry.



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