I'm currently running a phagocytic assay in which FITC-labeled E.coli bioparticles are being engulfed by lymphocytes. When running my control samples, however, the particles appear so bright that they bled into the FL2 and FL3 channels in the flow cytometer, when they should just appear in the FL1. I've tried quenching them in 1.2mg/ml of Trypan Blue immediately before analysis, but it isn't effective as some literature seems to make it out to be. Does anyone have any suggestions on how to remedy this situation? Possible voltage or compensation settings that worked for you?
I presume you don't want to alter the dose of particles, but can you prepare a new batch of particles with a lower loading of FITC?
That would useful, but I'm unsure how feasible that would be. I ordered my particles pre-labeled from a company (Molecular Probes) and didn't see any options on different intensities of coating for the different dyes available. That combined with the fact that these specific particles aren't uniformly stained, like FITC polystyrene beads, for instance, gives slight problems as well. It's definitely worth looking into, however.
Hey Marc what instrument are you working with? What are the control samples that you mention above? What is the MFI of the "test" samples versus the controls?
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