Dear colleagues Can anyone recommend a method that works for them for quenching FITC-tagged agent when bound to the external cell surface but not when the agent is internalised by the cell. Trypan blue and crystal violet come to mind - does anyone have this up and running - can you give method/recipes? I assume the quenching agent has to be present in the final cell suspension to be run on the cytometer - is it just a matter of resuspending the washed FITC-labelled cells in a quencher solution (in PBS?) and running them immediately - or are there finite times/temperatures involved. Is it true that only Merck trypan blue works? Has anyone compared trypan blue to crystal violet - and which came out best? Are there other quenchers which may be better? Do any of these quenchers significantly contaminate the cytometer tubing - how do you clean after running? Thanks in advance for any help Robin Barclay SNBTS, Edinburgh
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