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View additional product information for SYTO™ Green Fluorescent Nucleic Acid Stains - FAQs (S32703, S7575, S7574, S7573, S7572, S7578, S7556, S34854, S7576, S34855, S7559)
22 product FAQs found
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
PBS can be acceptable in some workflows, but we do not recommend assuming it is always the best-performing buffer for all three probes. Some applications may perform better in phosphate-free buffers, so we suggest optimizing as needed. When a starting point is needed, we recommend Live Cell Imaging Solution (LCIS; Cat. No. A59688DJ).
We recommend using these stains as relative imaging reagents within a controlled workflow rather than as absolute measurements of total RNA content. SYTO RNASelect Green reagent has been used in quantitative RNA content assay workflows, but any quantitative application should be carefully calibrated and ideally confirmed with an orthogonal RNA quantitation method.
No. We recommend thinking of SYTO RNASelect Red, SYTO RNASelect Green, and SYTO 14 reagent as RNA-associated stains rather than sequence-targeted probes.
Yes. We suggest these dyes can be used for automated segmentation and machine-learning analysis, depending on the workflow. SYTO 14 reagent has strong Cell Painting precedent, and SYTO RNASelect Red reagent has shown utility in high-content imaging workflows that rely on segmentation-oriented nucleolar analysis.
We recommend optimizing the staining protocol by increasing the dye concentration stepwise, confirming that the stock is fully homogeneous, extending the incubation time within the documented range, and verifying that the fixation method matches the dye. For SYTO RNASelect Green reagent, we do not recommend PFA fixation because it can flatten the expected staining pattern.
We recommend first reducing the dye concentration and shortening the incubation time. We also suggest adding a wash step, comparing your culture medium with a low-background imaging buffer, and confirming that the stock solution was fully dissolved before dilution.
We recommend using plastic tubes for dilutions because SYTO dyes can adhere to glass, which can lower the effective concentration and increase variability.
Yes. Both SYTO RNASelect Green and SYTO 14 reagents are stored at -25°C to -5°C. Because these products contain 100% DMSO and are frozen, we recommend letting the vial warm to room temperature before opening it and then briefly centrifuging it before use.
Yes, but we recommend choosing the RNA dye around the existing reporter. For example, if GFP is already present, we usually suggest SYTO RNASelect Red reagent to keep the green channel available. During assay setup, we also recommend checking the RFP channel for any bleed-through from the SYTO dye.
We do not recommend this for routine imaging. Because SYTO RNASelect Red, SYTO RNASelect Green, and SYTO 14 reagents all bind RNA-associated structures, combining them can create interpretation and competition issues without adding useful biological context.
Yes. We suggest confirming that the fluorophores are spectrally separated in your imaging setup before finalizing the assay.
Yes. We generally expect good spectral separation, but we recommend matching DAPI or Hoechst to your live-cell or fixed-cell workflow.
We do not consider permeabilization necessary for dye entry. In the workflows we tested, 0.5% Triton X-100 was compatible with downstream staining and had minimal impact on RNA signal.
Yes. We have found that SYTO RNASelect Green, SYTO 14, and SYTO RNASelect Red reagents can all be used after fixation. We do recommend keeping in mind that some staining features can change after fixation. For example, mitochondrial puncta seen with SYTO RNASelect Green reagent may disappear after fixation. SYTO RNASelect Red and SYTO 14 reagents are also compatible with permeabilization before labeling.
Yes. We have found that SYTO RNASelect Green, SYTO 14, and SYTO RNASelect Red reagent can all be used to label live cells before fixation. Of the three dyes, only SYTO RNASelect Red and SYTO 14 reagents are compatible with a permeabilization step.
Yes, but the fixation chemistry matters. We recommend SYTO RNASelect Red and SYTO 14 reagents for workflows that use PFA fixation followed by immunofluorescence. SYTO RNASelect Green reagent uses methanol fixation, which can work well for some antibodies but may disrupt epitopes for others.
We do not recommend treating direct intensity comparisons across SYTO 14, SYTO RNASelect Green, and SYTO RNASelect Red reagents as a simple measure of RNA abundance. These dyes differ in spectra, formulation, brightness, and workflow, so any cross-dye comparison should be considered assay-specific and calibration-dependent.
We usually recommend SYTO RNASelect Red reagent because it keeps the green channel available for GFP or FITC-labeled probes and antibodies.
We recommend SYTO 14 reagent if you need the closest fit to a Cell Painting workflow because it has the strongest direct literature association with Cell Painting. We suggest using SYTO RNASelect Red reagent as an RNA-focused or channel-specific alternative rather than as a direct replacement for a Cell Painting protocol. We also offer seminar and application note materials describing modified Cell Painting workflows that open channels for additional probes or antibodies.
We recommend SYTO RNASelect Green reagent when methanol fixation is acceptable and a defined RNA-selective workflow is desired. SYTO 14 reagent can also be imaged in FITC/GFP filter sets.
We recommend starting with SYTO RNASelect Red reagent or SYTO 14 if your workflow requires 4% PFA fixation. We recommend cold methanol fixation for SYTO RNASelect Green reagent over PFA fixation.