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Tech Tip: Cell Surface Stains for Live & Fixed Cells

Membrane staining is very useful in defining cell boundaries in multicolor imaging studies. Researchers are ever looking for suitable dyes to use under various viability, fixation and experimental staining conditions dictated by their particular workflow or application.

Biotium offers a large selection of highly fluorescent and photostable stains to visualize cell boundaries and morphology in multicolor staining experiments. To select the cell surface stain that is right for you, read about the different types of stains below, and consult the selection table at the end of the page.

#1  The original CellBrite™ dyes: Lipophilic carbocyanine dyes (DiO, DiI, DiD and DiR) that label membranes in a wide variety of live or fixed cells. Simple staining procedure, the dyes can be added directly to normal culture media of suspension or adherent cell cultures. Staining is non-toxic and stable with very little dye transfer between cells making them suitable for cell labeling, tracking mixed cell populations, and cell fusion studies.

While labeling with lipophilic carbocyanine dyes is very stable, the cellular localization of staining changes over time in live cells. Immediately after staining, the dyes primarily label the plasma membrane, but become internalized by endocytosis over the course of hours to a day, so staining becomes localized to intracellular vesicles and lysosomes. For long term imaging of cell morphology, our ViaFluor® SE dyes may be a more suitable option. See our Tech Tip: Using ViaFluor® SE Stains for Cell Tracing and Co-Culture.

Cells can be fixed with formaldehyde (PFA) before or after CellBrite™ staining, but staining has poor tolerance for permeabilization or methanol fixation, and cannot be used on FFPE sections, so they are not compatible with some immunofluorescence (IF) protocols. Good results can be obtained when cells are PFA-fixed and permeabilized with detergent prior to staining, see our Tech Tip: Combining Lipophilic Membrane Dyes with Immunofluorescence. CellBrite™ dyes are ready-to-use dye delivery solutions, with colors ranging from blue to near-IR. The CellBrite™ NIR dyes are suitable for small animal imaging. These dyes do not stain bacteria or yeast.

#2 Other lipophilic carbocyanine dyes for membrane labeling: Biotium also offers a selection of stand-alone carbocyanine dyes. Neuro-DiO, Neuro-DiI, and dilinoleyl dyes have structural features designed to make the probes diffuse faster in cell membranes. Near-infrared DiR is useful for small animal imaging.

#3  The fixable cell surface stains – CellBrite™ Fix and MemBrite™ Fix: Developed to overcome some of the shortcomings of the original CellBrite™ dyes, these novel covalent stains are compatible with fixation and permeabilization for multicolor IF staining. Staining is non-toxic and homogenous, but unlike the original CellBrite™ dyes and lectins, these dyes cannot be used on pre-fixed cells.

CellBrite™ Fix Membrane Stains are fluorogenic reactive membrane dyes that rapidly accumulate at the plasma membrane and react covalently with membrane proteins for stable labeling. Staining is single-step, no wash and rapid, taking only 15 minutes. CellBrite™ Fix can be used to stain mammalian cells, yeast, and bacteria.

MemBrite™ Fix Cell Surface Stains do not bind lipids, but covalently label cell surface proteins. Staining is two-step, requires washing, but offers a more extensive choice of dye colors than CellBrite™ Fix. MemBrite™ Fix also can be used to stain gram positive bacteria and yeast.

#4  Lectin conjugates for cell surface staining WGA, Con A and PNA: Conjugated to our bright and photostable CF® dyes, lectins label glycoproteins on the surface of live or fixed cells. Lectin staining is compatible with PFA, solvent fixation, or FFPE tissue. In pre-fixed and permeabilized cells, lectins stain both cell surface and organelles in the secretory pathway.

WGA and Con A lectins are widely used for cell surface staining in mammalian cells, and are also useful bacterial Gram stains and yeast stains. Unlike CellBrite™ or MemBrite™ stains, lectin staining may be cell-type dependent. We offer WGA and Con A with a wide selection of bright and photostable CF® dyes. Due to its better solubility, WGA is a more convenient option than Con A for general cell surface staining. We also offer PNA lectin conjugates, which stain less commonly expressed glycoproteins and have more specialized applications.

Find the right stain for your application or workflow: To compare and choose the right stain for your application, see the table below.

For more details, also see our Membrane & Cell Surface Stains Comparison or download the Membrane & Cell Surface Stains Selection Guide. Also see our Tech Tip: Five Steps to Successful Cell Membrane Staining and Imaging.