Hi Michael,

Could you expand on your methodology? Why would you fix the bugs before adding them to tissue culture? If you want to kill them can you not heat kill?

Also is it to monitor them? Can you not use a fluorescent tag?

Carla

Dear Michael,

I understood almost your approach and maybe fixing a certain 'thing' to unravel how it works is a good approach but do not forget that after PFA fixative ....you have at first control whether your target is changed or not? How did you do that? At second what is the final concentration of your solution-containing PFA-fixed 'things'?... However macrophages phagocytes foreign ententities smaller than...micrometer fixed of not.

Hi Carla,

I am also going to do heat killing as another method. I want to fix the bacteria so that when I add them with the macrophages they will still be recognized through TLR4 but the macrophages cannot kill them. They are tagged with GFP so I can fix the macrophages afterwards and use microscopy to observe the bacteria inside.

Michael-

This is a question that I have considered myself on many occasions. You are, of course, heavily modifying the outer appearance of the bacteria by fixing the cells before giving them to the macrophages. I would hypothesize that the fixed bacteria would look more like live than would heat-killed bacteria. Depending on the method of heat-killing you could end up causing a great deal of lysis. 

I would recommend filtering your fixed bacteria through a filter that will allow single cells to pass but remove clumps because the cross-linking activity of PFA might create clumps of bacteria that will affect your results. 

Another method to consider is the use of ionizing radiation to destroy your bacteria's DNA. This may be the least damaging to the normal function of the cells outer membrane. 

You need to determine if the surface protein of the bacteria is not grossly altered as to affect the characteristic features of the organism after  fixing with PFA. If altered this can affect recognition and also alter the pattern of phagocytosis for qualitative assessment,except if the assessment  is not  needed in your result.

Difficult to answer.....

We are working with fluorochrome-expressing E.coli and PFA Fixation has an influence on fluorochrome detection via FACS (especially GFP). Moreover, fixed E.coli are commercially available in some kits to quantify phagocytosis. We do obeserve differences between living and fixed E.coli in very late steps of the phagocytic process (killing and destruction of bacteria), but Ingestion and transport to the phagolysosome seems not to be hampered. Please consider that Fixation will lower the amount of shedded LPS which could interfere with TLR4 signalling.

Good luck!

Thank you everyone for your input. Some interesting discussion. Stephan, the fact that there is less LPS shedding may actually benefit my experiment because I want the macrophage to interact with the bacterium to initiate TLR4 signalling.