The apoptosis, necrosis and cell viability assays are designed to stain dissociated cells in culture and have not been validated for organ culture. Annexin V staining of early chicken and mammalian embryos in culture has been reported in the scientific literature. For staining of living tissues, the specimen would need to be thin enough to allow exposure of the cells to the 36 kDa Annexin V protein. Also, damage to cell membranes from dissection or sectioning of tissues could result in high background staining.
No. Fixation, freezing, sectioning, or dissociation of tissues can affect the PS on the outer leaflet and compromise membrane integrity. Both Annexin V and Ethidium Homodimer III rely upon the presence of intact membranes in healthy cells to accurately distinguish healthy cells from apoptotic or necrotic cells. To detect apoptosis in fixed cells and tissues we recommend our TUNEL kits. Currently we are not aware of a fluorescent probe that specifically detects necrotic cells in fixed tissues. Necrotic cells in tissue sections are identified based on morphological criteria.
Yes, cells can be fixed with formaldehyde after staining. Because Annexin V staining is dependent on calcium, all buffers used for washing and fixation should contain 1.25 mM CaCl2. Fixation may increase the background signal from Ethidium Homodimer III. Wash the cells several times to remove unbound Ethidium Homodimer III before fixation.
Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.
One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.
Coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.
Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature or with an ice pack. These products may thaw without affecting product performance. When you receive the product, place it under the recommended storage conditions.
CF® Dye Annexin V Conjugates can be used to stain the surface of apoptotic cells. The human anticoagulant Annexin V is a 35-36 kDa calcium-dependent phospholipid-binding protein with high affinity for phosphatidylserine (PS). In normal viable cells, PS is located on the inner leaflet of the cytoplasmic membrane. In apoptotic cells, however, PS is translocated from the inner to the outer leaflet of the plasma membrane, where it can be detected by fluorescently labeled Annexin V.
Azide-free for no-wash, real-time live cell imaging in culture medium
Fast & simple detection of phosphatidylserine on apoptotic cells
Choice of 11 CF® dye colors with superior brightness & photostability
For real-time live cell imaging, fluorescence microscopy, or flow cytometry
Preservative Free for Real-Time, Live Cell Imaging
Annexin V conjugates typically are supplied as stock solutions with azide as a preservative for end-point staining assays in Annexin V binding buffer. Our azide-free CF® Dye Annexin V Conjugates are supplied as lyophilized solids with no azide or other preservatives that might be incompatible with live cell imaging. After reconstitution in buffer, the conjugates can be added to cell culture medium for no-wash, real-time live cell imaging. Our Mini Syringe Filters are convenient for small volume sterile filtration of azide-free Annexin V stock solutions or other aqueous solutions for use in cell culture.
See Annexin V staining in real time:
Superior CF® Dyes
Biotium’s next-generation CF® dyes were designed to be highly water-soluble with advantages in brightness and photostability compared to Alexa Fluor®, DyLight®, and other fluorescent dyes. Learn more about CF® Dyes.
Kits to covalently label dead cells, allowing cells with permeable plasma membranes to be excluded from analysis in flow cytometry. A wide variety of dye options for standard flow, spectral flow, and fluorescence microscopy.