The Live-or-Dye™ fixable viability stains, including Live-or-Dye NucFix™ Red, are dead cell stains that are amine-reactive. This property of the dyes makes staining compatible with fixation, but challenging in multi-cell layer systems such as 3D, Matrigel® or organoid cultures. As the dyes are reactive, the majority of the staining would be restricted to the outer/ exposed cell layer/s. Longer incubation times to allow the dye to penetrate deeper in to the cell layers would generally not be helpful as the labeling reaction is short-lived (15-30 min).

See our recommendations for dyes suitable for 3D cultures or organoids.

For fixable nuclear dead cell stains, other options to consider would be NucSpot® Far-Red. Designed to be an improved alternative to the more common dead cell stain 7-AAD, the dye is fixable to a degree. Although generally more suitable for flow cytometry as its spectral properties are not appropriate for standard fluorescence microscopy, imaging using a confocal may be better as the Ex/Em can be more appropriately matched. We would also recommend imaging soon after fixation and not storing the sample for extended periods post-fixation as staining would deteriorate over time.

The viability dye PMA may be another possibility. PMA, a photoreactive version of propidium iodide (PI), is generally used for viability PCR of bacteria. However, it can be used with mammalian cells (based on internal customer feedback) as a fixable dead cell stain for flow cytometry. PMA is not fluorescent until it binds DNA. Subsequent exposure to bright light activates the dye and causes it to photo-crosslink to DNA, making it fixable. Incubation of the dye with cells would need to be done in the dark, and the excess dye washed away before exposing to light for photo-crosslinking. Optimization for light exposure sources (LED lights can be use to avoid heat) and light intensity (bright enough to crosslink the dye without photobleaching; generally close proximity to a bright LED or halogen lamp is required, room light is not sufficient) may be required.

Lipophilic carbocyanine dyes like CellBrite® Cytoplasmic Membrane Dyes may stain tissue slices or organotypic cultures, but due to their lipophilicity, they probably will not penetrate through multiple cell layers in a thick tissue slice. The dyes have been used for cell tracing in organoids and labeling hydrogel cultures. However, for this application, usually the cells are labeled before they are placed in organoid culture or embedded in hydrogel.

This class of dyes have been widely used for neuronal tracing in organisms, tissue slices, Drosophila embryos, and chicken embryos by microinjection or spot-labeling with crystals of solid dye. Neuronal tracing also can be done in formaldehyde-fixed tissues or embryos.

See our Tech Tip: Researching Applications for Membrane Dyes for more information and selected references.

Matrigel is a registered trademark of Corning Inc.

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. CellBrite® Fix reacts with plates coated with poly-L-lysine, collagen, gelatin, or other proteins, resulting in high background. The dyes tend to have high background on uncoated cell culture surfaces as well. Imaging cells by confocal microscopy can reduce interference from out-of-plane background fluorescence.

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 and Gram-positive bacteria. But unlike original CellBrite® Dyes and lectins, CellBrite® Fix and MemBrite® Fix cannot be used on cells that are already fixed. While we do not expect MemBrite® Fix stains to react with poly-L-lysine coated surfaces, we have seen high background with these types of plates and with uncoated cell culture surfaces. To circumvent this issue, we recommend imaging cells by confocal microscopy to reduce out-of-plane background fluorescence. The stains will react with surfaces treated with collagen, gelatin, fibronectin, or other extracellular matrix protein coatings.

GlycoLiner™ Cell Surface Glycoprotein Labeling Kits are designed for covalent labeling of glycoproteins on the surface of live cells. Like CellBrite^® Fix and MemBrite^® Fix, staining can withstand fixation and permeabilization. GlycoLiner™ also offers less cytoplasmic background in dead cells than CellBrite^® Fix and MemBrite^® Fix, allowing easier imaging of cell surfaces. GlycoLiner™ can also be used to stain yeast and Gram-positive bacteria. GlycoLiner™ is not expected to react with poly-L-lysine-coated surfaces, however, glycosylated matrix components like collagen or proteoglycans in complex matrices like Matrigel® may be labeled.

CytoLiner™ Fixed Cell Membrane Stains are novel lipophilic fluorescent dyes developed for selective plasma membrane staining in fixed cells and are suitable for downstream immunofluorescence staining protocols. Please note, staining with CytoLiner™ Dyes is not compatible with cells fixed using solvents like methanol or acetone, or with paraffin-embedded samples, because these treatments will remove the lipids from cells, which are required for CytoLiner™ staining. For co-staining with antibodies, we recommend staining with CytoLiner™ Dye first, then blocking with 2% fish gelatin in PBS, followed by antibody incubation in the same buffer. Blocking with BSA or serum is not recommended. CytoLiner™ staining is compatible with poly-L-lysine coated culture surfaces and Transwell® permeable supports. For best results, confocal microscopy is recommended for imaging fluorescent staining of Transwell® supports to avoid background from the filter material.

To select a dye that's right for your application, see our Membrane and Cell Surface Stains Comparison, or download our Membrane & Surface Stains Brochure.

Biotium ships all antibodies (primary, secondary and conjugates) at room temperature. We guarantee their quality and performance under these conditions based upon our stability testing. Antibodies were subjected to accelerated stability testing by storing them at various temperatures (4°C, room temperature, or 37°C) for 1 week to mimic simulated shipping conditions and tested in immunostaining experiments. All antibodies showed the expected brightness and specificity, even after storage at sub-optimal temperatures for a week or longer. You can also download our Product Storage Statement here.

In line with our goal to be more environmentally friendly by reducing the use of excess packaging, and lowering shipping costs for our customers, products that have passed our stability testing are shipped at room temperature.

Once you have received the antibody vial, please follow the long-term storage instructions on the product information (PI) sheet.