In order to outline a procedure which would save time for using three fluorescence dyes staining together, after testing each dye individually, all the three dyes were originally considered to be mixed altogether and tested on the cells for simultaneously staining. However, because of its chemical nature to interact with the anitibodies on CD45-PECy7 and EpCAM-FITC, Hoechst33258 is unable to stain cells together with CD45-PECy7 and EpCAM-FITC. Therefore in the mixed dyes staining test, Hoechst33258 was used first to stain the nucleus of cells for 10 minutes, and then the mixture of CD45-PECy7 and EpCAM-FITC was introduced simultaneously for the next 25 minutes of staining.
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