Cells were transfected with a pC1-EGFP-CAP-H construct in 4-well glass bottom dishes (LabTek). 48 hours post-transfection, SiR-DNA probe (1:2000, Spirochrome) was added to cells to visualize DNA, in L-15 CO2-independent medium (GIBCO). FRAP experiments were performed using a confocal microscope (Leica Microsystems) with a 63x oil immersion objective using the LAS-AF FRAP-Wizard. Images were taken by accumulating 4 frames at a scanning speed of 1000 Hz. Photobleaching was performed by 6 times illuminating the selected region at 100-fold laser power (488 nm Laser). 60 post-bleaching images were taken with 10 s intervals. Analysis of images was done using an in-house ImageJ macro. A mask on the DNA signal was created and the EGFP-CAP-H signal was normalized to the DNA signal.
L 15 co2 independent medium
Manufactured by Thermo Fisher Scientific
L-15 CO2-independent medium is a cell culture medium designed for the growth and maintenance of a variety of cell types in the absence of a CO2-enriched atmosphere. It is a balanced salt solution that supports cell proliferation and survival without the need for a CO2 incubator.
Automatically generated - may contain errors
3 protocols using l 15 co2 independent medium
1
Visualizing EGFP-CAP-H Dynamics by FRAP
2
Live Cell Imaging in L-15 Medium
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Cells were plated on 8-well glass-bottom dished (LabTek). Cells were imaged using a Delatavision deconvolution microscope (Applied Precision) equipped with a heated chamber and cultured in L-15 CO2-independent medium (GIBCO). Images were acquired every 4 min using a 20× (NA 0.25) objective. Z-stacks were acquired with 2.5-μm intervals. Images were processed using Softworx (Applied Precision), Fiji image software and Adobe Photoshop and Illustrator CS6.
3
Time-lapse Imaging of DNA in Live Cells
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Cells were plated on 8-well glass-bottom dishes (LabTek) and cultured in L-15 CO 2 independent medium (GIBCO). Cells were imaged using a Deltavision deconvolution microscope (Applied Precision) equipped with a heat chamber. For DNA visualization, 250nM SiR-DNA (Spirochrome) was added 2 hours before imaging. Images were acquired every five minutes using a 20x (0.25 NA) objective. Z stacks were acquired with 2 mm intervals. Images were analyzed and processed using Softworx (Applied Precision) and ImageJ.
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