Hello, I have been using Cell Mask to stain the plasma membrane of bEnd.3 cells. However, I find that Cell Mask fluorescence can be seen throughout the cell. I incubate with cell mask (1x manufacturer concentration) for 8 min, then PBS wash, then fix with 3.8% formaldehyde for 10 min at room temp, then dapi. I think the fixation step may be permeabilizing the cell membrane, since I've read on many message boards that formaldehyde fixation can permeabilize cells. Some people I've asked believe DAPI is cell impermeant, and therefore my cells are likely permeabilized since DAP nuclear signal is observed. However, I have read that DAPI can permeate across live cells, though less efficiently. Any suggestions on trouble shooting. I'm thinking 2% formaldehyde for 5 min, and maybe excluding DAPI in case it does something to the CellMask.
Dear Iason,
Permeability of the plasma membrane depends on many factors. How confident are you of cells that you are using are not permeable before you fix them? Given that your CellMask incubation is prior to fixation, then it is unlikely that your problem is due to subsequent fixation. However be aware that the fixative may also generate autofluorescence. In my experience if the cells are healthy and able to maintain membrane potential at physiological levels, fixation does not alter the permeability of the PM significantly and to increase permeability I use Triton-x. I suspect that the cells you are using are not very healthy and already leaky before you fix them and therefore CellMask is labelling entire cell, unless it is entirely due to autofluorescence/fixative and not due to CellMask labeling. I suggest that you use or borrow some other cell type that you are confident that is in good health and try CellMask on them and see if you can label the plasma membrane only. In the past there were similar questions that I have answered here at RG, so please feel free to browse through my technical report listed below.
Best wishes,
Refik Kanjhan
Technical Report Research Methods and Technical Considerations: Answers to ov...
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