Hello, I have to give infection with Mycobacterium tuberculosis to my cell culture and then proceed with cell surface staining for FACS. The problem with M.tb is I have to fix them with PFA before bringing it out of BSL3. So can I fix the infected mammalian cells with PFA, and then proceed for cell surface stain for FACS, or fixing has to be done after staining only?
Hello Mr. Naveen Baid ,
Take 3 set of cells .
1. Simply, stain cells first and then Fix them ( as we do routinely ) .
2. Prefix cell and stain them later on .
Now, run FACS.
Compare both group controls and prefixed - post fixed results.
I hope, you will get your optimised protocol and best results.
Best regards,
Babu
You can fix the cells prior to surface staining but just be aware that the PFA may alter the epitope of your surface protein and potentially would not be recognized by the antibody. You should test this by fixing some cells that are not infected with the bacteria along with cells not fixed/not infected and stain them to determine if PFA caused a loss of signal in your target surface proteins.
Yes, in addition to Tom's comments, try methanol fixing uninfected cells as an alternative trial experiment. Get institutional biosafety committee approval for these procedures.
Indeed the answers provided by Tom and Godwin will help you a lot. You can fix the cells to prior FACS staining by using PFA or some company company like BD available fixation buffers. In case of PFA protocol , you may find some some differences. But the BD reagent for fixation is good as such without hindering the epitope as said by Tom. One thing important here is cells have to be preferably be fixed before staining.
Hello Mr. Naveen Baid ,
Take 3 set of cells .
1. Simply, stain cells first and then Fix them ( as we do routinely ) .
2. Prefix cell and stain them later on .
Now, run FACS.
Compare both group controls and prefixed - post fixed results.
I hope, you will get your optimised protocol and best results.
Best regards,
Babu
PFA will give haphazard signal in flow by denaturing cell surface marker before Ab binding.
In addition to previous comments, you can try adding Ab in culture before bacterial infection (if you have short incubation time and if you have enough Ab plus less culture media). Ab will bind over time of infection.
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