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Disruption of the Actin Cytoskeleton with Cytochalasin D. U2OS human osteosarcoma cells were transduced with 'Cellular Lights' Actin-RFP (Actin-red fluorescent protein) and Cellular Lights MAP4-GFP (MAP4-green fluorescent protein) and imaged the next day. Cells were washed in HBSS and incubated with 1 ug/ml of the DNA stain Hoechst 33342 for 10 minutes then washed with HBSS. Cytochalasin D, an inhibitor of actin microfilaments, was applied to the bath at a final concentration of 10 uM.
Cells were imaged on a Deltavision core microscope with a 40X objective, 1.4 NA. Fluorescence was detected using the appropriate filters (DAPI/FITC/TRITC). Images were taken every 20 seconds for 30 minutes. In this video, microtubules appear green, actin microfilaments appear red, and the nucleus is stained blue. Note the disruption of actin filaments (red) whereas despite the change in cell shape microtubules are still clearly visible (green). The time series was deconvolved using standard software. Scale bar = 20 microns.
Invitrogen, Eugene OR
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