What do you mean the best way? The sample is E.coli? what is the antibody tagging? And what are the tags on the antibodies? If it's just one or 2 colors, compensation should be minimal.

Depending on your flow machine, most new ones have a compensation algorithm built in. Do single color staining of your sample and define your negative and positive regions and run the program.

If your machine can't run compensation automatically... depending on how many colors of your panel, it can be difficult.

You need to do the single staining of each fluorescent-conjugated antibodies, which used in this experiment, for your sample, and then compensate by following the manufacture of flow cytometry device.

Hi,

The best way is to have controls for all the samples. Like the normal unstained and unstained for treated one. Then you can have single cell stain for every fluorochrome which you are using for your experiment.

More important, check the correct voltage for E. coli, which you should use for getting good signals. Also, have a proper gating strategy before you jump to acquire the samples.

These small things can help you a lot to get best results and in a better way.

Best,

Hamid

Here is a nice video tutorial on compensation: https://youtu.be/Kh1WDqrke0Q

The principles of compensation remain the same irrespective of the fluorescence source, cell type, or application.

All the best!