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CELLestial™ Dyes & Kits FAQs
How do the ROS/RNS dyes work?
Nitric Oxide (Red dye): The strategy for fluorescence-based detection of NO employs an o-phenylenediamine scaffold, which in the presence of NO and air oxidizes to the corresponding aryl triazole. The electronic differences between the electron-rich diamine and electron-poor triazole groups provide a robust switch for NO detection. A crucial feature contributing to the success of this particular diamine-based probe is its high selectivity for NO under aerated conditions, as the fluorescent triazole product is not formed by reaction with superoxide, hydrogen peroxide, or peroxynitrite. We have observed poorer selectivity with diaminofluorescein (DAF) compared with our Red NO detection reagent, noting reactivity of DAF with both NO and peroxynitrite. Our probe is designed to form an insoluble precipitate upon reaction with nitric oxide. This prevents it from leaching out of cells, a problem encountered with DAF and its reaction product.
ROS (Green dye): The oxidation of the ROS detection reagent produces a green fluorescent compound. The detection reagent reacts with a broad range of reactive oxygen species, as indicated in our product literature. Reaction with such free radicals does not generate a fluorescent free radical, simply an oxidized dye that is fluorescent. Selectivity of the assay is simply determined by testing in the presence and absence of N-acetyl cysteine (NAC), a broad-spectrum ROS inhibitor. By using other inhibitors, it is even possible to classify the type of ROS that is generating the signal. This is illustrated in our workflow diagram below, following the green column in the diagram. Our protocol involves both ROS inducer and inhibitor controls to assure investigators of the selectivity of the assay.
Superoxide (Orange dye): The oxidation of the Superoxide detection reagent produces an orange fluorescent compound. The detection reagent reacts with superoxide but not other reactive oxygen species. Reaction with superoxide does not generate a fluorescent free radical, only an oxidized dye that is fluorescent. Selectivity of the assay is simply determined by testing in the presence and absence of a superoxide generator, such as actinomycin A as well as a superoxide scavenger, such as Tiron.
Does Dihydrorhodamine 123 (Ultra Pure), Prod. No. ENZ-52302, detect superoxide?
No, this fluorophore is not typically used for superoxide detection. We recommend instead Superoxide Detection Kit for microscopy and flow cytometry, ENZ-51012.
For the ROS/RNS detection kit, ENZ-51001 can samples be stored after the final wash buffer step in the protocol, and do the microscopy at a later date?
No, this is a vital stain. Live cells must be read immediately per the manual.
Do the cell cycle analysis dye change the growth characteristics of the cells under study?
All nuclear stains intercalate into DNA, stalling topoisomerase, and thus the answer is yes.
For cell cycle analysis, is it necessary to wash the cells after staining?
Both Nuclear-ID™ Green Cell Cycle and Red Cell Cycle kits do not require a wash step.
Does the apoptosis/necrosis kit work in fixed cells?
GFP-Certified™ Apoptosis/Necrosis assay must be performed with live cells. Fixing afterwards is OK.
How does the apoptosis/necrosis kit distinguish between early and late stage apoptosis?
In the early stages of apoptosis, phosphatidylserine (PS) flips from the inside to the outside of the cellular membrane with the cellular membrane still intact. Enzo Gold-Annexin V has very high affinity to PS therefore immediately detects early apoptotic cells.
Necrosis detection reagent is a cellular impermeable dye that cannot penetrate healthy cell membrane. In the late stages of apoptosis or during necrosis, the cellular membrane becomes compromised and allows Necrosis detection reagent to penetrate the cells.
What are the Lyso-ID® and Mito-ID® organelle dyes conjugated to, or to what (and how) they bind specifically to the organelles.
Conventional fluorescent probes for highlighting lysosomal compartments become trapped within lysosomes upon their protonation. These dyes fail to be retained within the organelles once vesicular pH values begin to rise due to concentration of certain drugs within the vacuolar lumen. We speculated that such probes would accumulate to differing extents based upon differences in the ionization constants of the different substituent groups and the overall membrane lipid permeability of the probes. Lyso-ID® Red and Lyso-ID® Green are novel fluorescent probes that selectively sequester in acidic organelles by a mechanism that likely involves protonation and retention within the organelles. However, by careful selection of titratable groups on the probe, staining has been extended into the vacuoles of cells pretreated with weakly basic, cell-permeant compounds, such as the anti-malarial drug chloroquine. This is critical to making the probe useful in drug toxicity screening and related applications.
Accordingly to the chemiosmotic theory, the mitochondrial respiratory chain converts the redox free energy made available by “down-hill” electron transfer from reduced substrates to O2, in active H+ translocation from the matrix toward the intermembrane space. This generates a transmembrane electrochemical potential ΔμH+ contributed mainly by an electrical component (ΔΨ, negative inside) and by a ΔpH (alkaline inside). The proton motive force so generated (estimated to rise up to 200-250 mV) does not collapse because of the low H+-conductance of the inner mitochondrial membrane and is instead utilized to drive up-hill reactions, first of all the ATP synthesis by the H+-ATP synthase . Most mitochondrial stains take advantage of the transmembrane electrochemical potential to concentrate in this organelle. However, any environmental assault on a cell tends to lead to loss of the transmembrane electrochemical potential, causing dye dissipation. The inner mitochondrial membrane is highly specialized: it contains a high proportion of the anionic “double” phospholipid cardiolipin, whose specific function is still to be completely elucidated. Our dyes are targeted to this stable feature of the mitochondria rather than to the transient membrane potential. Consequently, Mito-ID® Red and Mito-ID® Green highlights mitochondria regardless of energetic status. Mitochondria can even be fixed and permeabilized.
What is actually being stained in the nucleolus when using the Total Nucleolar/Nuclear Green/Red kit?
We use an RNA binding dye and this has been validated in the test tube. However, in the context of live cells we cannot rule out the possibility that we are binding to nucleoli proteins such as some of the silver staining methods do.
What is the Kd and quantum yield of FluoForte™ Calcium Assay kit?
Kd is estimated to be 389 nM (22°C, pH 7.0 –7.5)
Maximum QY is 0.18 at pH 7.2
