i think three time washing of the treated or infected cells with 37c PBS is enough to remove the non ingested bacterial cells, besides you can compare this with permeabilized cells with triton or any other permeablizer agent to check any differences between them, it is really hard to get 100% clean out the non invaded bacteria or beads as there will be some bacteria within the engulfed fold (vacules) of phagocytes but not truly ingested, however you can check this by TEM but it costs too much.

Hi,

for washing is always good to use warm media. In case it will not harm your experiment you can add also EDTA (0.2M) and BSA to the wash buffer.

Good luck

Michael

Gentamicin 30 min kill  the extracellular  bacteria :)

You can use a stain that changes color when comes in macrophage  I guess Rhodamin 123.  so The beads out of the macrophage membrane are stained different colour from the beads inside the macrohage.

It's quite difficult to wash away the extracellular particles, because they will often be bound quite tightly to multiple receptors on the cell surface. If you just need to differentiate between intracellular and extracellular particles, then there are two general approaches:

In both cases you let the phagocytosis happen, and then cool the cells down and add azide to stop further phagocytosis, before quenching/counterstaining.

Thank you everyone for all the great advice! I am currently not using flow to analyze phagocytosis- just imaging with fluorescent microscope.