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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

CellBrite® Cytoplasmic Membrane Dyes are ready-to-use dye delivery solutions for lipophilic carbocyanine dyes (DiO, DiI, DiD, and DiR) which label membranes in a wide variety of live or fixed cells. The dyes are available with colors ranging from blue to near-IR and can be added directly to the standard 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. The CellBrite® NIR dyes and near-infrared DiR are suitable for small animal imaging. The Original CellBrite® and CellBrite® NIR dyes do not stain bacteria or yeast.

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 labeling of membranes, see CellBrite® Steady (below). For fixable, long-term imaging of cell morphology, our ViaFluor® SE dyes, especially ViaFluor® 405 and ViaFluor® 488, 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. Still, staining has poor tolerance for permeabilization or methanol fixation, and cannot be used on FFPE sections and is therefore 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.

Biotium also offers a selection of stand-alone carbocyanine dyes, including formulations in vegetable oil optimized for microinjection. Neuro-DiO, Neuro-DiI, and dilinoleyl dyes have structural features designed to make the probes diffuse faster in cell membranes.

#2 CellBrite® Steady

Unlike other membrane dyes that are eliminated from the cell surface by endocytosis, CellBrite® Steady equilibrates between the surface and intracellular compartments, and is designed for long-term live cell staining. Cells labeled with CellBrite® Steady retain both surface and intracellular staining over time in culture so live cells can be imaged for 48 hours or longer. Intracellular staining becomes more prominent with longer incubation times.  CellBrite® Steady Enhancer, an optional reagent provided in the kit, reduces or eliminates intracellular staining and improves selective imaging of cell outlines and boundaries over several hours or days.

Membrane staining with CellBrite® Steady Dyes is retained immediately after fixation with formaldehyde but does not tolerate methanol or detergent. If cells are stored after formaldehyde fixation, the dyes will redistribute to stain cytoplasmic structures relatively quickly. CellBrite® Steady Enhancer cannot be used in a workflow that includes cell fixation.

CellBrite® Steady Dyes readily transfer between cells and are not recommended for cell tracking, co-culture, or transplantation studies. Our stable, non-toxic ViaFluor® SE Cell Proliferation Kits can be used to covalently label cells for long-term tracking by microscopy or flow cytometry. See our Tech Tip: Using ViaFluor® SE Stains for Cell Tracing and Co-Culture to learn more.

#3  The fixable cell surface stains – CellBrite® Fix and MemBrite® Fix

Biotium developed these novel covalent stains to be compatible with fixation and permeabilization for multicolor IF staining. Staining is non-toxic and homogenous, but unlike the original CellBrite® dyes and lectins (below), these dyes cannot be used on pre-fixed cells. If cells are cultured after staining, the dyes will be internalized over time by endocytosis, so we recommend fixing shortly after labeling.

CellBrite® Fix Membrane Stains are reactive membrane dyes that rapidly accumulate at the plasma membrane and react covalently with membrane proteins for stable labeling. The single-step staining protocol is rapid, takes only 15 min, and washing is not required. 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 a two-step procedure that requires washing but offers a more extensive choice of dye colors than CellBrite® Fix, including STORM-compatible MemBrite® Fix-ST dyes. 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. Lectin staining is cell-type dependent, unlike CellBrite® or MemBrite® stains. While lectins are commonly used for tissue staining, their localization is highly tissue-dependent, so they may or may not stain cell boundaries in your tissue of interest.

We offer WGA and Con A conjugates with a wide selection of bright and photostable CF® dyes. WGA is a more convenient option than Con A for general cell surface staining due to its better solubility. 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.

Sample/WorkflowCellBrite®CellBrite® SteadyCellBrite® FixMemBrite® FixCF® Dye WGA & ConA
Stain liveYesYesYesYesYes
Stain for real time live cell imagingYesYesNoNoNo
Stain live, then fix with PFAYesNoYesYesYes
Stain live, then fix with PFA/methanol & permeabilizeNoNoYesYesYes
Fix (PFA), then stainYesNoNoNoYes
Fix (methanol), then stainNoNoNoNoYes
Stain bacterial cellsNoNoYesYesYes
Stain yeastNoNoYesYesYes

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.