live cell confocal microscopy - protocol please? (Feb/14/2006 )

Would someone please send me the protocol for live cell confocal microscopy?

Thanks in advance.

In our lab we have been doing extensive long term fluorescence imaging with both CHO cells and BSC1 cells on a spinning disk. So if you want to do imaging over 2-3 days the best thing to do is to seed cells onto 22-24mm coverslips. This depends on the chamber you are using. We use an open version of a rose chamber (used for cutting and microinjections)with 2 coverslips placed above each other with FC43 and HEPES buffered media placed in between. We use red hydrophobic pen to place lines at top of coverslip which are lined up, so the media is kept within the gap and prevented from leaking out. Mineral oil is then overlaid to prevent media evaporating.

Other tips are

keep media phenol red free, i reacts with light and is harnful to cell

we use oxyrase, it absorbs dissolved oxygen in media and prevents photobleaching and formation of free radicals which can lead to cells becoming stressed under fluorescence

For more information papers by alexey khodjakov, conly rieder and edward salmon are useful sources of information for how to go about imaging correctly

Thank you very much