Using FACS analysis, is it possible to obtain dot plot having cells with unattached-bacteria and cells with attached-bacteria after infection?
Hello Kamlesh,
Of course, you can detect the fluorescent bacteria attached on eukaryotic cells for analysis by flow cytometer. It depends on which fluorescence you are going to use and also the Flow cytometer machine, you are going to use, have the suitable lasers and channels for the fluorescence chromophore. Besides, even you can sort and collect your cells if you use a FACS ( fluorescence-activated cell sorting) machine. But I don't know the bonds between cells and bacteria are weak or strong. If it is a week bond, then it could be broken during the flow cytometer preparation protocol. Because the cells for flow cytometer must be very homogenous and not attached each other.
I hope my answer will help you
Best
Cigdem
Dear Cigdem,
Thank you very much for your time for writing an answer. Your reply is useful for me.
So if the bond between cells and bacteria is not strong then I may not see any fluorescence; this is because sample processing for FACS may detach bacteria from the cells. It is hard to distinguish between bacteria infected cells and bacteria attached cells with FACS. Do you have any suggestion?
Regards
Kamlesh
Just a question Kamlesh to clarify concepts, your bacteria infect the cells? or they are just able to get attached without entering?
In my mind the answer is important, specially if both phenomena may happen.
Dear Rafael,
Thank you for the input.
Bacteria that I am using indeed infect the cells.
Then FACS should be useful to discriminate quite well between infected and non infected cells (as Cigdem pointed out). You have to optimize the assay and be sure that the cells are infected but not yet dead. Anyways it would be also relatively easy to look for dead cells in a parallel FACS run with the same sample but using apoptosis (?) or another suitable marker. Even if the machine allows discriminating 3 "colors" you may have all the data from 3 markers in a single run.
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