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Identification of cellular pathways of "type 1," Th17 T cells, and TNF- and inducible nitric oxide synthase-producing dendritic cells in autoimmune inflammation through pharmacogenomic study of cyclosporine A in psoriasis. Citations & References

  • Authors: Haider AS, Lowes MA, Suarez-Farinas M, Zaba LC, Cardinale I, Khatcherian A, Novitskaya I, Wittkowski KM, Krueger JG
  • Journal: J Immunol
  • PubMed ID: 18209089

I want a metabolic label to be incorporated into proteins or other cellular structures by adding the reagent to the media of cultured cells or in vivo injection. What are my options? Product FAQ

Answer

There are three types of metabolic labeling reagents available:


  • Click chemistry (azide-alkyne)
  • Staudinger ligation (azide-phosphine)
  • Photoreactive amino acids

  • The incorporation of azide-modified biomolecules permits eventual detection or crosslinking using alkyne or phosphine reagents. The incorporation of alkyne-modified biomolecules allows eventual detection or crosslinking using azide-modified reagents. The incorporation of either EdU or EU requires that the cells/tissues possess a pyrimidine salvage pathway. 

    Answer Id:: E15684

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    I got a low signal in media when I used a Gaussia/Cypridina/Renilla Luciferase Glow/Luciferase Flash assay. What happened? Product FAQ

    Answer

    Here are possible causes and solutions:

    - Insufficient luciferase accumulation in media: Incubate cells for a longer time.
    - Low luciferase expression: Use less media per well during the experiment;.Use a different promoter or growth conditions to improve expression;.Increase the integration time on the instrument;.Scale-up the volume of sample and reagent per well.
    - Treatment interfered with cellular secretory pathway: Transfect cells with a plasmid for constitutive expression of Luciferase (i.e., with pTK or pCMV promoter); Determine if luciferase actively expresses in media without treatment. Add treatment; Determine if there is a corresponding drop in luciferase activity from the constitutively expressed plasmid.

    Answer Id:: E15658

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    Deciphering the mechanism of HNE-induced apoptosis in cultured murine cortical neurons: transcriptional responses and cellular pathways. Citations & References

    • Authors: Peng ZF, Koh CH, Li QT, Manikandan J, Melendez AJ, Tang SY, Halliwell B, Cheung NS
    • Journal: Neuropharmacology
    • PubMed ID: 17889908

    User Guide: TaqMan Gene Expression Assays - TaqMan Array Plates Manual / Product Insert

    • Version: H
    Catalog #

    Two pathways for insulin metabolism in adipocytes. Citations & References

    • Authors: Duckworth WC, Hamel FG, Peavy DE
    • Journal: Biochim Biophys Acta
    • PubMed ID: 9332452
    Catalog # D113(Discontinued)

    What cellular processes can be analyzed with a flow cytometer? Product FAQ

    Answer

    -Calcium flux: Each of the Oregon Green calcium indicators binds intracellular calcium with increasing affinity, providing a sensitivity range to match many applications. Oregon Green probes emit green fluorescence at resting levels of Ca2+ and increase their fluorescence intensity 14-fold with increasing Ca2+ concentration. The cell-permeant formulation (Cat. No. O6807) can be loaded in cell media and is compatible with flow cytometry.
    -Rhodamine-based calcium indicators comprise a range of probes for large or small changes in Ca2+ concentration. They exhibit a 50-fold increase in fluorescence upon calcium binding and offer a range of wavelengths that can be used in conjunction with GFP or green-fluorescent dyes for multiplexing. Rhod-2, AM (Cat. No. R1245MP), in particular, localizes to mitochondria and can be used with flow cytometry.
    -Membrane potential: A distinctive feature of the early stages of apoptosis is the disruption of the mitochondria, including changes in membrane and redox potential. We offer a range of products specifically designed to assay mitochondrial membrane potential in live cells by flow cytometry, with minimal disruption of cellular function. The MitoProbe family of mitochondrial stains (Cat. Nos. M34150, M34151, and M34152) provide quick, easy, and reliable flow cytometric detection of the loss of mitochondrial membrane potential that occurs during apoptosis. MitoTracker dyes (Cat. Nos. M7510 and M7512) are membrane potential-dependent probes for staining mitochondria in live cells. The staining pattern of MitoTracker dyes is retained throughout subsequent flow cytometry immunocytochemistry, DNA end labeling, in situ hybridization, or counterstaining steps. The Mitochondrial Permeability Transition Pore Assay (Cat. No. M34153) provides a more direct method of measuring mitochondrial permeability transition pore opening than assays relying on mitochondrial membrane potential alone. The mitochondrial permeability transition pore (MPTP) is a non-specific channel formed by components from the inner and outer mitochondrial membranes, and appears to be involved in the release of mitochondrial components during cell death.
    -Phagocytosis: In phagocytosis, cells internalize particulate matter such as microorganisms, and this process is important for immune responses and during the clearance of apoptotic cells. Probes for studying phagocytosis include BioParticles indicators—bacteria and yeast labeled with fluorescent dyes.
    -Tracking phagocytosis using a quench/wash-based assay can report on simple uptake, or a pH indicator can be used to monitor stages in the pathway. We have no-wash assays labeled with pHrodo Red or Green (Cat. Nos. A10010, P35361, P35364, P35365, P35366, and P35367) and no-wash assays for whole blood (Cat. Nos. A10025, A10026, P35381, and P35382), all suitable for flow cytometry.
    -pH changes: Sensitive pH determinations can be made in a physiological range using either fluorescent intensity or ratiometric measurements. pHrodo dyes (Cat. Nos. P35373 and P35372) provide signal intensity modulation from pH 2 to pH 9 and with a choice of fluorescent wavelengths. Tracking internalization of fluorescent dextran is a routine method for analyzing pH changes in cellular compartments. Dextran conjugates of pHrodo dyes (Cat. Nos. P35368 and P10361) provide the most complete solution by allowing discrimination of vesicles from early endosomes to lysosomes, with no quench or wash required.
    -Reactive oxygen species: Cells that are environmentally stressed usually contain greatly increased levels of reactive oxygen species (ROS). CellROX reagents are fluorogenic probes developed for the detection and quantitation of ROS in live cells. These cell-permeant reagents are non-fluorescent or very weakly fluorescent in the reduced state; however, when oxidized, they become brightly fluorescent and remain localized within the cell. We offer CellROX Green (Cat. No. C10492), CellROX Orange (Cat. No. C10493), and CellROX Deep Red (Cat. No. C10491) Assay Kits validated for flow cytometry.

    Answer Id:: E14827

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    Amyloid β induces cellular relocalization and production of agrin and glypican-1 Citations & References

    • Authors: Timmer, NM; van Horssen, J; Otte-Holler, I; Wilhelmus, MMM; David, G; van Beers, J; de Waal, RMW; Verbeek, MM
    • Journal: BRAIN RESEARCH

    If the protein/enzyme cannot be isolated in pure form, is the assay still valid? Product FAQ

    Answer

    Not all proteins/enzymes can be isolated in pure form. They may require accessory proteins or chaperonins for activity or require that their hydrophobic domains are covered with lipids, such as with membrane proteins/enzymes. Even proteins/enzymes isolated by affinity purification may co-elute with other cellular components that do not dissociate easily. 

    Validity is established by understanding what else is bound to the protein/enzyme of interest and how those other components can affect the assay. Ideally, the material should be analyzed by native and/or SDS-PAGE as well as other analytical methods to determine total protein concentration and the concentrations of other components (e.g., lipid content, polysaccharide content, metal ion content, etc.).

    If your sample is hydrophobic, it may bind to any lipids associated with the protein/enzyme even though these never enter the active or binding site. Also, hydrophobic components and interactions may cause the compound to bind to the surfaces of microplate wells or cuvettes. A direct assay may help with such samples. 

    If the presence or absence of an impurity affects the activity or binding properties of a protein/enzyme, different lots of the protein may vary in the amount/ratios of these impurities may result in variations in the final results. 

    Answer Id:: E15847

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    Signaling pathways mediating β(3)-adrenergic receptor-induced production of interleukin-6 in adipocytes Citations & References

    • Authors: Tchivileva, IE; Tan, KS; Gambarian, M; Nackley, AG; Medvedev, AV; Romanov, S; Flood, PM; Maixner, W; Makarov, SS; Diatchenko, L
    • Journal: MOLECULAR IMMUNOLOGY

    Nifuroxazide inhibits survival of multiple myeloma cells by directly inhibiting STAT3. Citations & References

    • Authors: Nelson EA, Walker SR, Kepich A, Gashin LB, Hideshima T, Ikeda H, Chauhan D, Anderson KC, Frank DA
    • Journal: Blood
    • PubMed ID: 18824601

    What is the mechanism for microRNA- and siRNA-mediated RNA interference? Product FAQ

    Answer

    RNA interference has evolved to describe the process for repressing or silencing gene expression. Whether the small RNA that triggers the event is of an endogenous (miRNA) or exogenous (siRNA/shRNA) origin, the mechanism is similar. The RNA interference pathway (RNAi) is a process in which a double-stranded RNA (dsRNA) triggers the degradation of a homologous mRNA. An enzyme called Dicer cleaves the dsRNA into small duplexes of 21-25 nucleotides. The siRNAs are combined into a multi-protein complex called RISC (RNA-induced silencing complex), and the RISC is guided to mRNAs that have perfect homology with the siRNAs, targeting them for sequence-specific degradation. MicroRNAs are thought to be processed by the same cellular mechanism as the RNAi pathway including Dicer and the RISC complex. RNAi has become a tool to study gene function by blocking the expression of specific mRNAs and generating “knockdowns” (reverse genetics).

    Answer Id:: E2761

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    Human lung fibroblasts express interleukin-6 in response to signaling after mast cell contact. Citations & References

    • Authors: Fitzgerald SM; Lee SA; Hall HK; Chi DS; Krishnaswamy G
    • Journal: American Journal of Respiratory Cell and Molecular Biology
    Catalog #

    Gene profiling and signaling pathways of Candida albicans keratitis Citations & References

    • Authors: Yuan, XY; Mitchell, BM; Wilhelmus, KR
    • Journal: Molecular Vision

    Nifuroxazide inhibits survival of multiple myeloma cells by directly inhibiting STAT3 Citations & References

    • Authors: Nelson, EA; Walker, SR; Kepich, A; Gashin, LB; Hideshima, T; Ikeda, H; Chauhan, D; Anderson, KC; Frank, DA
    • Journal: BLOOD
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