CellBrite® Cytoplasmic Membrane Stains are lipophilic dyes for simple, non-toxic, stable labeling of membranes in live or fixed cells. Cells can be fixed with formaldehyde before or after CellBrite® staining. But the staining has poor tolerance for permeabilization or methanol fixation, so CellBrite® staining is not easily combined with intracellular immunofluorescence (IF) staining. The dyes also do not stain bacteria or yeast. CellBrite® NIR dyes are CellBrite® dyes with near-infrared fluorescence compatible with small animal NIR imaging systems.
CellBrite® Fix and MemBrite® Fix stains were developed to overcome some of these shortcomings. They are novel covalent stains that can be fixed and permeabilized for IF staining. CellBrite® Fix Membrane Stains are fluorogenic reactive membrane dyes that rapidly accumulate at the plasma membrane. When they incorporate into lipids, they become fluorescent, and at the same time react covalently with membrane proteins for stable labeling. Staining takes only 15 minutes in a single step with no wash. CellBrite® Fix stains mammalian cells, yeast, and bacteria.
MemBrite® Fix Cell Surface Stains do not bind lipids, but label cell surface proteins. MemBrite® Fix requires a two-step staining protocol with washing, but offers a more extensive choice of dye colors than CellBrite® Fix. MemBrite® Fix also can be used to stain yeast. But unlike original CellBrite® dyes and lectins, CellBrite® Fix and MemBrite® Fix cannot be used on cells that are already fixed.
Biotium’s CellBrite® Cytoplasmic Membrane Dyes are dye delivery solutions that can be added directly to normal culture media to uniformly label suspended or adherent cells in culture. The PKH dyes are structurally related dyes for cell membrane labeling. But unlike CellBrite®, labeling with PKH dyes requires multiple steps and subjects cells to an iso-osmotic mannitol loading medium that can negatively affect cell membrane integrity and viability.
CellBrite® Fix dyes also feature rapid and simple labeling in isotonic buffer. Cells can be fixed and permeabilized after labeling with CellBrite® Fix, unlike original CellBrite® dyes or PKH dyes, which don’t tolerate detergent.
DiI (DiIC18(3); 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine) is a widely used lipophilic carbocyanine dye that labels cell membranes by inserting its two long (C18) hydrocarbon chains into the lipid bilayer. It has been extensively used for the antero- and retro-grade labeling of neurons.
Neuro-DiI (also available as a ready-to-use staining solution, CellBrite® Green) was developed by Biotium as an alternative to DiI. It has two additional t-butyl groups (one on each side of the dye) attached to the dye chromophore which introduce structural flexibility in the dye making it much more soluble in both organic solvents and in cell membranes. Improved dye solubility in the membranes enables rapid dispersion of the dye molecules, away from each other much more rapidly than regular DiI which has a relatively flat structure that promotes dye aggregation, and slows down dispersion of dye molecules. Neuro-DiI therefore results in more uniform, rapid and stable staining as compared to DiI.
FAST™ DiI uses a different strategy to solubilize the dye. Replacing the saturated C18 lipophilic chain of regular DiI with unsaturated linoleyl chains gives the dye molecules a bent structure, making them less likely to aggregate and thus disperse faster in the membrane.
Both Neuro-DiI and FAST™ DiI would diffuse faster than regular DiI in membranes, but a comparison of the relative diffusion rate between the two dyes has not been made. Neuro-DiI may have a potential advantage over FAST™ DiI. The t-butyl groups of Neuro-DiI render it more hydrophobic than FAST™ DiI, likely resulting in more stable membrane staining and make dye transfer between cells less likely.
FAST DiI is a trademark of Thermo Fisher Scientific.
MemBrite® Fix Cell Surface Staining Kits provide a convenient way to visualize cell boundaries in multicolor staining experiments. Staining is rapid and uniform, with a wide choice of colors. MemBrite® Fix dyes covalently label the surface of live cells. The staining withstands fixation and permeabilization for subsequent immunofluorescence staining.
Rapid, uniform live cell surface staining
Covalent labeling can be fixed & permeabilized for IF staining
Stain mammalian cells, yeast, or gram-positive bacteria
Choose from 12 bright & photostable dye colors from blue to near-IR
Dye options for super-resolution and other specialized imaging applications
Fixable Cell Surface Stains
MemBrite® Fix Stains are novel reactive fluorescent dye stains that react irreversibly with cell surface proteins, for staining that can withstand formaldehyde or alcohol fixation, and detergent permeabilization. Unlike lectins such as WGA, which bind specific targets that may vary between cell types, MemBrite® Fix dyes react widely with cell surface proteins. MemBrite® Fix staining is rapid and non-toxic to cells, and because MemBrite® Fix dyes are highly water soluble, they stain cells much more evenly than traditional lipophilic membrane dyes like DiO, DiI, PKH, Vybrant®, or CellMask™.
MemBrite® Fix staining kits also can be used to stain yeast and gram-positive bacteria, but not gram-negative bacteria. See our Cellular Stains Table for more information on how our dyes stain various organisms. MemBrite® Fix 405/430 has been validated for staining of extracellular vesicles (EVs) and exosomes.
MemBrite® Fix Staining Kits belong to Biotium’s line of novel cell surface stains that include CellBrite® Fix Membrane Stains. CellBrite® Fix Membrane Stains are fluorogenic dyes that rapidly accumulate in the plasma membrane, where they react covalently with the cell surface. CellBrite® Fix stains require only a single staining step compared to MemBrite® Fix staining, which is a two-step protocol. On the other hand, MemBrite® Fix dyes are available with a wider selection of colors. MemBrite® dyes do not associate with lipids in membranes, and consequently have lower cytoplasmic background after detergent permeabilization compared to CellBrite® Fix.
Selecting a MemBrite® Fix Dye
Several MemBrite® Fix dyes have been validated in super-resolution imaging applications or 2-photon microscopy. MemBrite® Fix-ST dyes are recommended for super-resolution imaging by STORM. MemBrite® Fix or MemBrite® Fix-ST dyes can be used for standard microscopy applications; however, MemBrite® Fix dyes are generally more photostable than MemBrite® Fix-ST dyes. See the MemBrite® Fix Product Table below for details.
Tips for Success
Note that MemBrite® Fix dye stain dead cell more intensely than live cells. With prolonged dye incubation, or if cells are cultured after staining, the dye also will be internalized by endocytosis, resulting in labeling of intracellular vesicles. Please see our Tech Tip: Five Steps for Success with Membrane and Surface Stains for tips on staining and imaging (step 5) with MemBrite® Fix.
MemBrite® Fix dyes are designed to be fixed shortly after staining. With prolonged dye incubation, or if cells are cultured after staining, the dyes will be internalized by endocytosis, resulting in labeling of intracellular vesicles. By 24 hours after staining, most of the dye will be localized inside the cell, not on the cell surface. For long-term visualization of cell boundaries in culture, we recommend our CellBrite® Steady Membrane Staining Kits. These kits include unique fluorescent membrane probes that retain cell surface staining in live cell cultures for 24 hours or longer. The kits also include an optional CellBrite® Steady Enhancer solution which masks intracellular signal for even greater specificity of cell boundaries.
1. Kit size based on 200 uL reaction volume, actual number of reactions will depends on staining volume used.
2. MemBrite™ Fix dyes are named for their Abs/Em maxima. Download the Product Information Sheet for spectra.
3. MemBrite® Fix-ST 681/698 dye is reported to have better performance in STORM imaging than MemBrite® Fix 680/700 dye.
Alexa Fluor & Texas Red are registered trademarks of Thermo Fisher Scientific; Cy Dye is a registered trademark of GE Healthcare; IRDye is a registered trademark of LI-COR Bioscience.
Vybrant is a registered trademark and CellMask is a trademark of Thermo Fisher Scientific.
Biotium’s CellBrite® NIR Cytoplasmic Membrane Dyes are ready-to-use dye stock solutions that can be added directly to normal culture media to uniformly label cell membranes with near-infrared lipophilic dyes.