Tech Tip: Five steps to successful cell membrane staining & imaging

Having trouble with cell surface staining?

Check out this tech tip on five steps for successful staining and imaging of cell membranes using Biotium’s cell surface stains. Featuring easy-to-use workflows and tips for classic lipophilic dyes, original CellBrite™ dyes, CellBrite™ Fix dyes, and MemBrite™ Fix dyes.

Tips for success with CellBrite™, CellBrite™ Fix, and MemBrite™ Fix membrane and cell surface stains

Membrane and cell surface stains are very useful for visualizing cell borders and morphology in multicolor staining of live or fixed cells. Biotium offers several options for cell surface imaging for different applications. To get the best results with our stains, check out these five tips for success:

1. Choose the right stain for live or fixed cells

Original CellBrite™ can be used to stain live or formaldehyde (PFA)-fixed and detergent permeabilized cells, see our Tech Tip: Combining Lipophilic Membrane Dyes with Immunofluorescence. However, they can’t be used on samples fixed with methanol or other solvents, or FFPE sections. CellBrite™ Fix, and MemBrite™ Fix can tolerate common fixation/permeabilization methods, but they must be used to stain live cells before fixation. If they are used on cells after they are fixed, these dyes will mainly stain the cytoplasm.

For more help choosing a dye (plus information on using these dyes in bacterial or yeast), see our Tech Tip: Cell Surface Stains for Live & Fixed Cells, or download our Membrane & Surface Stains Selection Guide.

2. Understand the workflow before you begin

Original CellBrite™ dyes, CellBrite™ Fix, and MemBrite™ Fix each uses a different labeling protocol. They also have different requirements for fixation and permeabilization. Being familiar with the labeling protocol before you begin is a key step for getting good staining.

To get complete, step-by-step protocols, download the product information sheets:

3. Use the right buffer for staining

Original CellBrite™ dyes can be added to cell culture medium with serum, or buffers like PBS. But CellBrite™ Fix and MemBrite™ Fix are chemically reactive compounds, so they have stricter buffer requirements. Make sure you’re using a compatible buffer to avoid interference with staining. When using CellBrite™ Fix or MemBrite™ Fix with adherent cells, our preferred buffer is HBSS with calcium and magnesium for maintaining cell adhesion and morphology. Using PBS without Ca2+/Mg2+ can cause some adherent cells to round up or detach.

4. Use the right fixative and mounting medium

Original CellBrite™ dyes are compatible with formaldehyde (PFA) fixation after staining. However, the stained cells can’t be treated with solvents like methanol, detergents, or mounting medium with glycerol. In contrast CellBrite™ Fix and MemBrite™ Fix can be fixed and permeabilized with any standard protocol, and mounted with antifade mounting medium like Biotium’s EverBrite™ Mounting Medium. See below for guidelines on fix/perm and mounting.

5. Know what to expect when imaging

Confocal vs. epifluorescence microscopy

If you have access to a confocal microscope, we recommend using it to image membrane staining for the best results. Confocal imaging screens out fluorescence from above and below the plane of focus, allowing very crisp imaging of cell boundaries. Compared to regular epifluorescence imaging, confocal is more sensitive and gives you more control over excitation power to limit photobleaching. Membrane dyes can be imaged with a regular epifluorescence microscope, but the images will be more diffuse because fluorescence from membranes above and below the cell borders will be captured.

Changes in dye localization over time in live cells

Original CellBrite™ dyes mainly stain the plasma membrane, even in fixed cells. The dyes themselves are very stable, and have been reported to stain live cells for weeks in culture. However, dye localization in live cells changes over time. If live cells are cultured after staining, the labeled membrane will be internalized, so staining will gradually change from cell surface to intracellular vesicles, usually becoming mostly intracellular after about 24 hours.

CellBrite™ Fix and MemBrite™ Fix localization will also change from cell surface to intracellular as membranes turn over, like original CellBrite™. Because they react with membrane proteins, staining with CellBrite™ Fix and MemBrite™ Fix is less stable than original CellBrite™. Fluorescence is usually detectable for up to 48 hours after staining.

For stable, long-term imaging of cell morphology, our ViaFluor® SE dyes may be a more suitable alternative than membrane stains. See our Tech Tip: Using ViaFluor® SE Stains for Cell Tracing and Co-Culture.

Staining of dead cells

CellBrite™ Fix and MemBrite™ Fix react irreversibly with cellular proteins. In live cells, this occurs on the cell surface, because the dyes can’t penetrate the membrane. But they do get inside dead cells, where there are many more targets for reaction. As a consequence, the dyes stain dead cells much more brightly than live cells. When imaging these stains, do not focus on very bright, rounded-up, or shrunken dead cells. Instead, adjust the plane of focus and imaging settings to detect the live cell membrane staining. The dead cell signal will likely be saturated under these settings. If the dead cell staining interferes with your imaging, try using high magnification and confocal imaging to exclude dead cells from the field of view. Or, try using one of our original CellBrite™ dyes, which do not show such dramatic differences in signal between live and dead cells.

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Find out more

See our Tech Tip: Cell Surface Stains for Live & Fixed Cells

Download our Membrane & Surface Stains Selection Guide

See CellBrite™ & MemBrite™ FAQs

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