NucView® Caspase-3 Enzyme Substrates
The substrates are very stable. Some users have reported performing time course assays with NucView® 488 Caspase-3 Substrate for 4-5 days.
NucView® Caspase-3 Substrates can be added to the cells at the start of the experiment or at the end. A major advantage of NucView® Caspase-3 Substrates compared to other apoptosis assays is that it can be used to monitor capase-3 activity in real time.
Yes. NucView® Caspase-3 Substrates are compatible with flow cytometers and other instruments with the appropriate excitation and emission settings.
Yes. We recommend a gentle fix in 2-4% paraformaldehyde for 10-15 minutes. Over-fixing can cause the signal to decrease. NucView® 488 staining can withstand permeabilization with 0.1% Triton X-100. Methanol fixation is not recommended.
NucView® Caspase-3 Substrates have not been validated by Biotium for live tissue staining. However, we have had feedback from a customer who successfully used NucView® 488 Caspase-3 Substrate for live zebrafish embryo staining. NucView® Caspase-3 Substrates cannot be used in fixed cells or tissues.
As with all fluorescence based probes, bleaching will occur over time. How long you can view NucView® staining under the microscope depends on several factors including the initial signal strength and the intensity of the excitation source. Some users have reported monitoring NucView® for 4-5 days. You can see a time-lapse video of NucView® staining over 24 hours here:
The NucView® 488 Assay Kit for Live Cells (30029) contains NucView® 488 substrate at 0.2 mM in DMSO and caspase-3 inhibitor Ac-DEVD-CHO. The final DMSO concentration in the kit assay can be fairly high, which is undesirable for sensitive cell types. Because of this, we offer NucView® 488 Caspase-3 Substrate at 1 mM in DMSO (10402) and at 1 mM in PBS (10403), so customers can control the amount of DMSO in their assay. Note that in non-DMSO sensitive cell types, adding DMSO during the substrate incubation can enhance NucView® staining.
DEVD-CHO is a reversible inhibitor and may not sufficiently block caspase-3 activity. Adding an irreversible inhibitor like DEVD-FMK at the beginning of the experiment (before or at the time of apoptosis induction) may more effectively inhibit caspase activity.
Like other caspase-3 substrates, NucView® 488 Caspase-3 Substrate is based on a DEVD sequence that also can be cleaved by caspase-7.
We currently offer NucView® in three dye colors: green (our original NucView® 488), blue (NucView® 405), and orange (NucView® 530). Other colors of NucView® are in development.
Our NucView® substrates detect cleavage by caspases 3 and 7. We do not currently have NucView® substrates for other caspases.
The tables below list primary cells types and immortalized cell lines reported to work with NucView® 488 in the published scientific literature. Click here for a PDF version of the tables along with the references.
|Primary cell type||Species|
|Alveolar epithelial cells||Mouse|
|Embryonic fibroblast (MEF)||Mouse|
|Idiopathic pulmonary fibrosis fibroblasts||Human|
|Immature B cells||Mouse|
|Kidney epithelial cells||Mouse|
|Lung microvascular endothelial cells||Human|
|Mammary epithelial cells (3-D cultures)||Mouse|
|SVZ neural progenitor cells||Rat|
|Pancreatic acinar cells||Mouse|
|Pancreatic beta cells||Rat|
|Pancreatic islet cells||Mouse|
|Pollen tubes||Field poppy(Papaver rhoeas)|
|Retinal pigmented epithelial cells||Human, mouse|
|Skin fibroblasts||Sand cat(Felis margarita)|
|Umbilical vein endothelial cells||Human|
|Vascular endothelial cells||Rat|
|Immortalized cell line||Species||Cell type|
|CCL-134||Human||IPF pulmonary fibroblast|
|FU-UR-1||Human||Renal cell carcinoma|
|Min 6||Mouse||Pancreatic insulinoma|
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.
Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, 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.
For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).
Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.
If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.
Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact firstname.lastname@example.org for assistance.
Chemical products with special stability considerations:
Ester compounds include the following:
• Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
• Acetoxymethyl esters (AM esters) such as our membrane-permeable ion indicator dyes
• Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes
Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.
A note on CF™ dye succinimidyl ester stability
Succinimidyl esters are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Heavily sulfonated dyes, such as the Alexa Fluor® dyes, DyLight® dyes and IRDyes® are particularly hygroscopic, worsening the hydrolysis problem. For example, the percent of active Alexa Fluor® 488 succinimidyl ester (SE) could be well below 50% by the time of application (according to the manufacturer’s product datasheet). In a number of Alexa Fluor® SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while in all of Biotium’s CF™ dyes the SE group is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF™ dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.
Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.
Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.
Coelenterazines and D-luciferin
Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.
Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.
Note that 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.
D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.
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.
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.