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Tech Tip: Five Steps to Successful Cell Membrane Staining & Imaging

Tips for success with Biotium’s line of CellBrite® and MemBrite® 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 cells 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, CellBrite® Steady, and MemBrite® Fix should only be used on live cells. If they are used on cells after they are fixed, these dyes will mainly stain the cytoplasm or intracellular structures.

If you are planning on imaging live cells for extended periods, we recommend CellBrite® Steady dyes which are designed to retain staining of cell surface membranes of living cells for up to several days in culture. Intracellular staining can be reduced or eliminated with the use of CellBrite® Steady Enhancer. CellBrite® Fix and MemBrite® Fix are covalent surface stains that are best used on live cells that will be fixed shortly after surface labeling for subsequent immunofluorescence staining.

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

Click to enlarge. *Lectin conjugates like ConA and WGA may be options for staining cells before or after MeOH fixation. See our Membrane and Cell Surface Stains Selection Guide for more information.

2. Understand the workflow before you begin

Original CellBrite® dyes, CellBrite® Fix, CellBrite® Steady, and MemBrite® Fix each use 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.

Click to enlarge. *We’ve had good results when PFA-fixed cells are permeabilized with 0.1% Triton® X-100 before staining with CellBrite®. See the product protocol for details.   † Optional step before imaging. Staining with antibodies or other dyes can be performed after fixation/permeabilization.

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

3. Use the right buffer for staining

Original CellBrite® and CellBrite® Steady 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.

Click to enlarge. *If your cells need a special buffer, contact Biotium technical support to ask about compatibility. Note that the use of the prestaining solution is required for efficient labeling with MemBrite® Fix.

4. Use the right fixative and mounting medium

Original CellBrite® and CellBrite® Steady dyes are compatible with formaldehyde (PFA) fixation. However, they can’t be used with solvents like methanol, detergents, or mounting medium with glycerol. In contrast CellBrite® Fix and MemBrite® Fix can be fixed, permeabilized, and mounted with any standard protocol, and mounted with antifade mounting medium like our EverBrite™ Mounting Medium. See below for guidelines on fix/perm and mounting.

Click to enlarge. * We’ve had good results when PFA-fixed cells are permeabilized with 0.1% Triton® X-100 before staining with original CellBrite® dyes. See the product information sheets for details.

5. Know what to expect when imaging

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 rapidly internalized, so staining will change from cell surface to intracellular vesicles, usually becoming mostly intracellular after a few hours.

CellBrite® Steady Dyes equilibrate between intracellular compartments and the plasma membrane. This allows the cells to retain surface staining as well as intracellular staining for several days. These kits also include CellBrite® Steady Enhancer as an optional reagent that can be used to mask intracellular fluorescence of CellBrite® Steady Dyes, providing more selective visualization of cell boundaries.

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,  CellBrite® Fix and MemBrite® Fix dyes may not be retained by cells for as long as 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 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. You may also try using one of our original CellBrite® dyes, which do not show such dramatic differences in signal between live and dead cells.

See products

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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