1. HeLa cells are easily transfected by most transfection reagents. It is our preference to use FuGENE 6 if given because of its simple protocol of use and low cytotoxicity. Following the recommendations from the developer, use Opti-MEM media without serum for transfection with FuGENE 6. The main recommendation from the developer of FuGENE 6 is that contact with plastic surfaces other than pipet tips should be avoided. Add FuGENE 6 directly into the volume of the Opti-MEM media.

2. For imaging, if available, use Leica confocal system TCS SP2 on inverted microscope Leica DM IRE equipped with HCX PL APO lbd.BL 63x 1.4NA oil objective and 125-mW Ar and 1-mW HeNe lasers.

3. I f cells used do not significantly change their shape during the course of imaging, the xt (line scan) mode can be used as it is less photo-damaging for the cells.

4. The lower resolution gives less detailed pictures, and the higher resolution slows the laser beam, which is more photo-damaging for the cell. In any experiment with the real-time dynamics of fluorescence, two applications are possible: to quantify the fluorescent signal or to make "pretty" pictures. For quantification, the use of low resolution is enough and much less damaging.

5. There are many liquid perfusion modules on the market. If the microscope one is using is equipped with one of them, add the compounds according to the instruction of module manufacturer. Also, if the microscope being used is equipped with CO2 control module, use Opti-MEM instead of L-15 media.

6. Differences in brightness between frames are better represented not in monochrome but by using a multicolor Lookup Table.

7. Leica format ".lei" is a stack of TIFF files. These files could be quantified by any analysis software such as freeware "ImageJ" (Waine Rasband, NIH,

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