Time-lapse imaging of fluorescently labelled cells in association with neurites <i>in vitro</i>.

<p><b>A–E</b>) Myelinating cultures generated from a mix of wild type/beta-actin mice were visualised using time-lapse microscopy (Nikon TE2000 (60×, 0.75NA) over 16 hr in 5 min intervals on DIV 27 after the addition of wild type neurospheres previously infected with lentivirus carrying dsRed/GFP gene and addition of cyto-GFP cells. Two cell types were followed over time, one that expressed DS red/cyto-GFP and the other cyto-GFP. <b>A–E</b>) Strongly positive green cells typical of cyto-GFP morphologically resembled oligodendrocytes in contact with neurite bundles. The membrane appears to ruffle and form flaps/bubbles (arrow). In addition, the soma changes its location with respect to the neurite processes, over time, by moving closer to the neurite bundle. <b>C–E</b>) Dynamic imaging over 7.5 hours of a dsred/GFP labelled cell (asterisk) which was engulfed by a cell resembling a microglial cell (yellow arrow). This fluorescence was very much weaker than the cells generated from the beta-actin cyto-GFP mouse. Time frames obtained with 40× magnification (long distance working lens) and without perfect focus. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030775#pone.0030775.s002" target="_blank">Video S2</a>. <b>F</b>) Immunostaining of a similar culture around the same time confirmed MBP expression. Representative video from 1 experiment, seen in duplicate.