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Volume 7 issue 12 December 2006
Spectral Shift of Fluorescent Dye FM4-64 Reveals Distinct Microenvironment of Nuclear Envelope in Living Cells
Tomasz Zal, M. Anna Zal, Carina Lotz, Craig J. Goergen, and Nicholas R.J. Gascoigne
Supplemental Material
Fig. S1. Visualization of NE in various cell types. Live cells were stained with FM4-64 and imaged with wide-field microscope as described. The long wavelength excited fluorescence of NE is in the red channel, short wavelength excited fluorescence is in the green channel. A, Human kidney epithelial line BOSC23. B, Mouse bone marrow-derived macrophages, (n) marks necrotic cells. C, Mouse T cell hybridoma A18.ZC.4Y expressing CD4-YFP in blue. D, Human T cell line Jurkat. Cells in mitosis are marked with (a). E, Dual excitation imaging of FM4-64 stained mouse bone marrow cells containing granulocytes with characteristically convolved NE. F, Live mouse splenocytes. Digital channel un-mixing was applied to A, B, C, D. No un-mixing was applied to E and F. Scale bars are 10 µm.
Figure S1 (.jpg)
Video 1. Kinetics of staining of intracellular structures upon addition of 20µM FM4-64 to mouse lymphocytes at 37o C. Time elapsed 7 min. No digital un-mixing was applied.
Video 1 (.mov)
Video 2. Kinetics of staining of intracellular structures at 25oC. Time elapsed 7 min. No digital un-mixing was applied.
Video 2 (.mov)
Video 3. FM4-64 staining of the B cell cancer line LK35 shown as a scan through a z-stack of images. In this movie the long wavelength excited NE fluorescence is green, other intracellular membranes and vesicles appear yellow and red (no digital un-mixing).
Video 3 (.mov)
Video 4. 3D reconstruction of a single cell NE in the LK35 B cell tumor stained with FM4-64. Staining was as described. Channels were separated into the NE (red) and endocytic vesicles (green) using the digital linear un-mixing algorithm. Images were then deconvolved by the constrained iterative algorithm and 3D rendering was done using the maximum intensity projection.
Video 4 (.mov)
Video 5. Loss of FM4-64 NE fluorescence marks T cell killing of a tumor cell. The data and description are the same as in Fig. 5.
Video 5 (.mov)
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