How do SYTO dyes bind to DNA?
The binding mode of SYTO nucleic acid stains is unknown. However, the behavior of these and related nucleic acid dyes suggests the following binding properties:
1.They appear to contact the solvent (suggested by sensitivity to salt, divalent cations, and in particular, SDS) and thus are likely to have contacts in the grooves.
2.All SYTO dyes appear to show some base selectivity and are thus likely to have minor groove contacts.
3.They can be removed from nucleic acid via ethanol precipitation; this characteristic is not shared by ethidium bromide and other intercalators. Likewise, the dyes are not removed from nucleic acid via butanol or chloroform extraction. These extraction methods do remove ethidium bromide from nucleic acid.
4. SYTO binding is not affected by nonionic detergents.
5. SYTO dyes are not quenched by BrdU, so they do not bind nucleic acids in precisely the same way as Hoechst 33342 and DAPI ((4′,6-diamidino-2-phenylindole).
SYBR Green I has shown little mutagenicity on frameshift indicator strains, indicating that it isn't likely to strongly intercalate.
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I want to study apoptosis using an Annexin V conjugate, but with adherent cells via microscopy instead of flow cytometry. Can this be done?
It has been done, but we don‘t recommend it. Both healthy cells and apoptotic cells possess phosphatidylserine on the cell surface, which can be detected with Annexin V, but apoptotic cells have significantly more of it. You can easily tell the difference between these two populations with flow cytometry, because flow cytometers are more sensitive and have a higher throughput. But with a microscope, you cannot always tell the difference, especially for adherent cells. Instead, for microscopy, we recommend a different technique, such as detecting caspases with CellEvent Caspase Detection Reagents.
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I trypsinized my adherent cells and labeled with annexin V, and now my flow data is showing a high percentage of apoptotic cells even for control, untreated cells. What is the problem?
Trypsinization or mechanical scraping of cells temporarily disrupts the plasma membrane, allowing annexin V to bind phosphatidylserine on the cytoplasmic surface of the cell membrane and thus leading to false positive staining. Allow the cells to recover for about 30 minutes in optimal cell culture conditions and medium after trypsinizing/scraping so that they can recover their membrane integrity before staining. For lightly adherent cell lines, such as HeLa and NIH 3T3, another option is to use non-enzyme treatments like Gibco Cell Dissociation Buffer (Cat. No. 13151014).
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Can I detect annexin V staining in an imaging assay?
Annexin V staining is not typically used in imaging experiments; it is a better reagent for flow cytometry analysis. All cells will stain to some extent, so it can be difficult to distinguish a relatively bright annexin V-stained cell from a dimmer non-apoptotic cell. Caspase activation, detected using our CellEvent Caspase 3/7 or Image-iT LIVE Caspase detection kits, is a better method for detecting apoptosis in an imaging assay.
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When should I stain adherent cells with annexin V for flow cytometric analysis? Before or after I trypsinize them?
Trypsinize first and then allow the cells to recover about 30 minutes in optimal cell culture conditions and medium before staining with annexin V conjugates. Trypsinization or mechanical scraping of cells temporarily disrupts the plasma membrane, allowing for annexin V to bind phosphatidylserine on the cytoplasmic surface of the cell membrane and thus leading to false positive staining. For lightly adherent cell lines such as HeLa and NIH 3T3, you could use a less harsh (non-enzymatic) dissociation product like Gibco Cell Dissociation Buffer (Cat. No. 13151014).
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
