How to check if it is intracellular infection? - (Sep/20/2008 )

I am infecting bacteria to cell lines. I am wondering whether it is a intracellular infection or just attachment on the cell surface. How can I check this? I mean, I was thinking of doing immunofluorescence and confocal but it will be on 2D image. How can I make sure it is actually entering the cell upon infection?

How about live cell imaging?

Any input will be more than welcome.

Cheers.

You could add antibiotics which do not enter the mammalian cells. This would kill off extracellular bacteria and only the ones inside would survive.

There are two ways to do this: confocal microscopy, which for sure will tell you if the bacteria are inside or outside the cell if you look at the different image layers by co localization with intracellular markers. The other way, that might take longer but it is very accurate is a double immunofluorescence as follows: do your infection, then wash and treat the culture with an antibiotic that does not penetrate the eukariotic cell wall, such as gentamicin. Then fix your cells with for instance paraformaldehyde. Add a first antibody against your bacteria, labelled with fluorescein for example. Then perform a cell permeabilization step using saponin, then add a second antibody against your bacteria and labelled with a different fluorescent dye, for instance Texas Red. When you look at the slides under the fluorescent microscopy the interpretation goes like this: those bacteria that are labelled green and red are outside the cell, whereas those labelled only red are inside the cell. The reason for this: the permeabilization step was performed after the first antibody (green) was added and therefore it can not recognize the intracellular bacteria. Of course, we have to run proper controls to check that everything is working as you expect. Good luck!