Annexin V Near IR CF® Dye Conjugates

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.

The exact chemical structures of CF® dyes are currently confidential but will be fully disclosed at a later stage when pending patents become granted. In general terms, the structure of a CF® dye may be divided into two parts: a) dye core structure (i.e. the aromatic ring skeleton that defines the dye’s color or absorption/emission wavelengths), and b) core structure-modifying elements. At present, CF® dyes bear the core structures of coumarin, pyrene, rhodamine or cyanine dyes. Blue fluorescent CF® dyes are based on coumarin or pyrene dye core structure, while green to near-IR CF® dyes are based on either cyanine or rhodamine dye core structures. Core structure-modifying elements refer to various chemical attachments to the core structure and are a key aspect of the CF® dye invention that makes CF® dyes superior to other commercial dyes.

Most CF® dyes carry 1-2 negative charges while a few cyanine-based near-IR CF® dyes carry 3-4 negative charges. However, the more negatively charged CF® dyes comprise unique structural features that shield the negative charges such that the biomolecules (such as antibodies) the dyes label do not lose specificity due to the excessive negative charges.

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.