I am staining B16 with hoechst dye and setting coculture with HaCat but the hoechst is also staining HaCat in coculture. Thus my purpose to stain cell to differetiate in coculture on the basic od labelling remain unsuccessful.
Since dyes are permeable to cells at 37 degree , do they also move out and maitain an equilibrium with the media?
if the cells do not proliferate too fast, you can try to combine different CelllTrace (CFSE analogous) or a cell trace with PKH26 (red) or PKH67 (green). They are membrane dyes that binds to lipides in cell membranes, they are diluted every time cell proliferate, but they can be easily detected for some days (depending on the proliferation rate). Check the detection possibilities at microscope and try to set a window where your cells do not lose the dye. Hope it helps, good luck!
1) Dyes can be used for this purpose, with the caveat of proliferation leading to dye dilution, as suggested by Michela. Also, to clarify, dyes such as CellTrace and CFSE are cell-permeant, amine-reactive dyes that bind to intracellular and cell surface proteins, not lipids (there are also lipophilic dyes). There are also CellTracker dyes with similar chemistry that may be better suited to excitation and detection sources of microscopes.
2) Regarding the possibility of dyes "leaking out" of the cells - this is highly unlikely with these amine reactive dyes, assuming the dyes are quenched after labeling with extraneous protein such as serum or BSA, and then washed before incubation with the other cells. Any dye in the cell is covalently bound to the proteins.
Use CFSE for labelling your cell of interest, it will not come out of the cells.
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