New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes.

摘要: The use of carboxyfluorescein diacetate succinimidyl ester (CFSE) to measure lymphocyte proliferation by flow cytometry has become one the most widely utilised assays for assessing responses. properties CFSE make it ideal such a task, covalently labelling cells with long-lived fluorescence high intensity and low variance minimal cell toxicity. No dye in last 20 years been capable replicating these respects. However, currently is limited following maximum 7 divisions not compatible ubiquitously available fluorescein conjugates or other fluorescent molecules spectral similar fluorescein, as EGFP. Here we characterise two new dyes measuring proliferation, Cell Trace Violet (CTV) Proliferation Dye eFluor 670 (CPD), which have different excitation emission spectra and, consequently, are conjugates. We found that while both CTV CPD can label intensity, variability toxicity makes them long-term tracking non-dividing lymphocytes vivo, offers possibly best alternative analysis divisions. also describe how intercellular transfer autofluorescence affect division resolution three conditions produce ultra-bright vivo studies allow up 11 be detected when using dyes.