Fluorescent Protein-Cell Labeling and Its Application in Time-Lapse Analysis of Hematopoietic Differentiation

Here, we present a computer-controlled time-lapse system for imaging of cultured hematopoietic cells labeled by the expression of different fluorescent proteins. First, we describe experiments to optimize the visualization of three green fluorescent protein variants (cyan-, green-, and yellow-enhanced fluorescent protein) and the red-fluorescent protein (DsRed) by standard wide-field fluorescence microscopy. Then, we describe procedures to best distinguish combinations of cells expressing these proteins using seven commercially available filter sets, based on the relative fluorescence intensities of the individual fluorescent proteins. Finally, we make recommendations about which of these filters to choose when working with specific fluorescent proteins.