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View additional product information for SYTO™ RNASelect™ Red - FAQs (S32710, S32711)
28 product FAQs found
There are some variations to the ultracentrifugation protocols, not based on the cell lines used so much as the experience in that particular lab, including what G-force they recommend, duration of ultra-centrifugation, straight sedimentation vs cushion vs sucrose gradients to obtain the top quality exosomes at reasonable yields. We often recommend protocols developed by Clothilde: C. Thery, S. Amigorena, G. Raposo and A. Clayton “Isolation and characterization of exosomes from cell culture supernatants and biological fluids.” Curr. Protoc. Cell. Biol., Chapter 3, Unit 3: 22, 2006.
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 have observed that SYTO RNASelect Red reagent fluorescence intensity can increase during live imaging under continued excitation. This can be mitigated with N-acetyl cysteine. For qualitative live imaging, this may be acceptable, but for quantitative comparisons we recommend standardizing illumination carefully or fixing the cells for endpoint imaging.
We recommend using plastic tubes for dilutions because SYTO dyes can adhere to glass, which can lower the effective concentration and increase variability.
Yes. SYTO RNASelect Red reagent can be stored from 2°C to 30°C. In practice, room-temperature storage up to 30°C is acceptable when the product is protected from light.
SYTO RNASelect Red reagent can be stored from 2°C to 30°C, but partial precipitation can occur during refrigerated storage. We recommend storing at room temperature or warming the vial and briefly centrifuging it before dilution if the product has been stored cold.
This usually reflects minor precipitation during refrigerated storage. We recommend warming the vial to room temperature and vortexing first. If crystals remain, warm the vial to about 37°C for about 30 minutes, vortex again, briefly centrifuge, and use the solution only after it becomes completely homogeneous. Partial precipitation can contribute to speckles or other staining artifacts.
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.
If methanol fixation is required for a specific assay, we recommend treating it as an optimization experiment because this workflow has not been tested.
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.
The aqueous SYTO RNASelect Red dye concentrate can be added directly to buffer or imaging solution. The following protocol was optimized with U2OS and SKBR3 cells and should be considered a guideline for other cell types. We recommend determining the optimal staining concentration within the working range of 0.1–10 µM. Prepare a 10 µM working solution of SYTO RNASelect Red imaging probe in PBS or Live Cell Imaging Solution (LCIS; Cat. No. A59688DJ) by making a 1:100 dilution from the stock solution. Fix the cells with 4% PFA for 20 minutes, wash three times with PBS, and optionally permeabilize with 0.5% Triton X-100 for 20 minutes. Then incubate the cells with the working solution for 30–60 minutes at room temperature, wash with additional PBS, and image.
The aqueous SYTO RNASelect Red dye concentrate can be added directly to cell culture media or live cell imaging solution. The following protocol was optimized with U2OS and SKBR3 cells and should be considered a guideline for other cell types. We recommend determining the optimal staining concentration within the working range of 0.1–10 µM. Prepare a 10 µM working solution of SYTO RNASelect Red imaging probe in an appropriate cell culture medium or Live Cell Imaging Solution (LCIS; Cat. No. A59688DJ) by making a 1:100 dilution from the stock solution. Incubate the working solution for 30–60 minutes at 37°C under standard tissue culture conditions, wash the cells with additional LCIS, and image under live-cell conditions. If fixation is needed after staining, remove the working solution, add 4% paraformaldehyde (PFA) for 20 minutes, wash three times with PBS, and optionally permeabilize with 0.5% Triton X-100 for 20 minutes before any additional counterstaining.