Hello Leyla Barry,

You should add trypan blue to the cell suspension at the right concentration after the phagocytosis assay has run its course. Too little trypan blue won't fully quench the extracellular beads, and too much of it might start to interfere with other aspects of the experiment or even enter cells. 

Trypan blue effectively penetrates and binds to the extracellular beads that have not been internalized. Since trypan blue cannot easily penetrate live cells or phagosomes, the beads that have been successfully internalized remain fluorescent. So, when you add trypan blue, it binds to the extracellular bead that are not internalised, and absorbs the light emitted by the fluorescent dye, reducing or eliminating the signal. Then you measure the remaining fluorescence after trypan blue treatment. It will quantify the number of internalized beads and assess the phagocytic activity of the cells. 

I feel you need to optimize the trypan blue concentration and incubation time to ensure efficient quenching of extracellular fluorescence without affecting intracellular fluorescence. 

Best,

the quenching is ineffective if using latex beads in our hands. we only use trypan blue quench when the particles are biological material, eg apoptotic cell debris or phptoreceptor outer segment fragments. the trypan blue will penetrate well into these if they are extracellular. also, quenching only works with green fluorescence. finally, the extent of fluorescence reduction may depend on your detection sensitivity (depends on your instrumentation/imaging).