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We used our fourth-generation lentiviral packaging system and one of our pLVX-lentiviral vectors to compare the virus production of the Lenti-X 293T Cell Line to that of two other commonly used HEK 293-based cell lines. All plots are gated on lymphocytes. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Excitation and emission profile most closely match BD Horizon V500 (V500-C) or AmCyan. & Raman, V. Noninvasive optical tracking of red fluorescent protein-expressing cancer cells in a model of metastatic breast cancer. Region - Euro A Inserting a cDNA in the MCS upstream of the tdTomato coding sequence joins your protein of interest to the N-terminus of the tag, and allows the fusion protein to be tracked and studied in transduced cells. Can be successfully unmixed from PE on a spectral cytometer. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. A copy of the ptdTomato-N1 Vector product License Agreement can be found by clicking here. Cells were washed and fixed with Fluorofix prior to analysis. Improved stability in fixative solutions. (A-B) Viral vectors expressing the two fluorescent proteins indicated were co-transfected in 293T cells alone or together. Citations: OriGene Technologies' Mouse monoclonal tdTomato antibody, Magnetic beads is a Mouse monoclonal antibody. So, if expression increases, it wont affect as many neighboring channels or exacerbate spreading error. Utilize this fluor on ubiquitously expressed markers or markers with varying levels of expression. red fluorescent protein, Creating new fluorescent probes for cell biology, The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford, Fluorescent indicators for simultaneous reporting of all four cell cycle phases, Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting, mScarlet: a bright monomeric red fluorescent protein for cellular imaging, Blue fluorescent protein derived from the mutated purple chromoprotein isolated from the sea anemone Stichodactyla haddoni, An improved monomeric infrared fluorescent protein for neuronal and tumour brain imaging, A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo, High-speed multiparameter photophysical analyses of fluorophore libraries, Minimum set of mutations needed to optimize cyan fluorescent proteins for live cell imaging, Proceedings of the National Academy of Sciences, Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%, An engineered monomeric Zoanthus sp. Because of their small size, multiple simple organic fluorophores can be conjugated to a single antibody. In this study, the performance of each fluorescent protein was characterized by examining these factors. Compensation beads capture species-specific antibodies conjugated to fluorophores and other types of reagents. Use our. A comparison of fourth- and third-generation lentiviral packaging systems . Avoid repeated freeze-thaws. red fluorescent protein. . Cookie Management. The tdTomato and ZsGreen1 reporters containing the DD had three- and six-fold greater fluorescence intensity respectively, than the versions without the DD, due to the latters increased background levels. In one xenograft mouse model of metastatic breast cancer, tdTomato was easily detected as deep as 1 cm below the surface, and extremely small lesions could be detected and tracked over time (Figure 2; Winnard, Kluth, and Raman 2006). Harvested viral supernatants were also analyzed by RT-PCR to quantify viral genome copies (qRT-PCR, Lenti-X qRT-PCR Titration Kit), ELISA to measure p24 (p24 ELISA, Lenti-X p24 Rapid Titer Kit), and by a rapid lentiviral detection method (Lenti-X GoStix). The small membrane-permeant ligand Shield1 binds to the DD and protects the entire fusion protein from proteasomal degradation. Autofluorescence contributed by cell type is also an important consideration since it will directly effect the sensitivity index of the fluorescent protein and, therefore, resolution of dim signals. But as these fluorescent proteins are detected in the same channel, only one can be utilized. Relatively high spillover from PerCP/Cyanine5.5, Alexa Fluor 647, and APC, which can compromise sensitivity when staining for co-expressed markers using these fluors. Use our. 24 hr later, the cells were stimulated with 10 M forskolin and simultaneously treated with 1 M Shield1. One of the brightest fluorophores, though this isnt always an advantage because PE/Cyanine5 heavily spills into a variety of other channels due to significant cross-beam excitation by other lasers,particularly the red laser. In addition, no other proteins exhibited significant spillover nor spreading into the GFP channel indicating that eGFP and any of the other proteins tested can be coexpressed without significant impact on resolution. PE/Cyanine7 may exhibit non-specific binding to monocytes and macrophages. Comparable compensation requirements into PE when compared to spectrally equivalent tandems. NUR-SULTAN - The regional commission on compensation to small and medium-sized enterprises affected by the tragic events of January has paid out 370 million tenge (US$854,000) to 221 business owners, reported the press service of the Almaty akimat (city administration). From these data, EGFP, Venus, mTagBFP2, TagRFP657 and mIFP emerge as the best candidates for creating a multicolor flow panel (Fig 2). Exposure time: 1 sec. However, the spreading error of PE/Fire 640 is less substantial using a system that has both a 488 nm blue laser and a 561 nm yellow/green laser. When used with theLenti-X Packaging Single Shots and the Lenti-X 293T Cell Line (Cat. As a result, only the reporter molecules expressed during promoter induction will contribute to the fluorescence signal, providing a considerably higher signal-to-noise ratio than that obtained with nondestabilized or constitutively destabilized reporter systems. Image from the RCSB PDB (rcsb.org) of PDB ID 1EYX Contreras-Martel, C. et al. With the 488 and 561 nm lasers occupied by the aforementioned proteins, we focused on mTagBFP2 for the 405 nm laser and TagRFP657, and mIFP for the 640 nm laser. The SSM was calculated using FlowJo and values were compared. ordersEU@takarabio.comtechEU@takarabio.com+33 139 046 880. (A) Representative confocal images of mK4 cells transduced with a viral vector encoding a single fluorescent protein. Privacy Policy Most natural fluorescent proteins cloned from different organisms function as dimers or tetramers, which can lead to aggregation of protein in the cell [10]. Care should be taken not to combine fluorescent proteins that heavily contribute to or have high spillover spread in other channels in use. Colored bars represent laser wavelengths (inset, left). Stemming from the development of GFP [13], a large number of fluorescent proteins have been discovered and generated, with origins outside GFPs Aequorea victoria [49]. Learn more about the unique advantages offered by each member among the various families of fluorophores and how to best use them in multicolor panels: Two of the most commonly used fluorophores in flow cytometry, R-phycoerythrin (R-PE) and allophycocyanin (APC), are phycobiliproteins originally derived from algae. Exceptional brightness: the brightest commercially available red fluorescent protein, Monomeric: Nonaggregating tandem dimer structure, FRET and other quantitative imaging techniques, Identify and select transfected cells with the IRES tdTomato vector, Transgenic micetdTomato is easily detectable in SCID mice, where fur is an additional impedance to fluorescence signal penetration (Winnard, Kluth, and Raman 2006). Terms of Use Ideally, these two fluors are not used to detect co-expressed markers. # 632180), in a 10 cm dish. 2004). The DD-tdTomato Reporter System includes the pDD-tdTomato Reporter vector and Shield1. No additional transfection reagent is needed because Lenti-X Packaging Single Shots (VSV-G) consist of pre-aliquoted, lyophilized, single tubes of Xfect Transfection Reagent premixed with an optimized formulation of VSV-G pseudotyped Lenti-X lentiviral packaging plasmids. From these results, we concluded that the fluorescent proteins with the highest SI values were the best candidates as reporters for low expression targets. Ideally, these two fluors are not used to detect co-expressed markers. Useful for detection in microscopy when utilizing an anti-FITC secondary antibody. A very potent donor with an extinction coefficient of 2,500,0 M-1cm-1 which makes for very potent tandem dyes. Use this bacterial expression vector as a source of the fluorescent protein gene. Certificates of Analysis and Kit Components Lists are located under the Documents tab. Use our. RBC lysed and washed human blood was stained with a multicolor panel with an intentionally high degree of spectral complexity and overlap. Human whole blood was stained with anti-CD19 conjugated to Spark Blue 550, anti-CD4 BV570, anti-CD38 PE, and anti-CD40 FTIC. IRES: internal ribosome entry sequence; FP1: fluorescent protein (AcGFP1, DsRed-Monomer, or mCherry); FP2: fluorescent protein (mCherry or ZsGreen1); MCS: multiple cloning site. To analyze promoter activity, a candidate inducer is added to the medium along with Shield1, which effectively stabilizes the reporter protein and allows it to accumulate. Tandem dye using PerCP as a donor and Cyanine5.5 as the acceptor fluorophore. The resulting lentivirus can then be used to transduce virtually any mammalian cell type. Specifically, generating reporters with combinations of three of these proteins would allow efficient measurements even at low-level expression. (A) Compensation values are indicated, however, plots are displayed without compensation applied. To analyze promoter activity, a candidate inducer is added to the medium along with Shield1, which binds to the DD tag and thereby stabilizes the reporter protein and allows it to accumulate. Relatively dim fluorophore, should be used for higher abundance targets. *Save favorites by clicking the star () in the top right corner of each page while you're logged in. Emits in the far red at 806 nm. Removal of Shield1, however, causes rapid degradation of the entire DD-fluorescent protein reporter. Imaging was conducted utilizing the parameters indicated in Table 1. DD-Fluorescent Protein promoter reporters provide a much greater fold increase in signal intensity than traditional fluorescent protein reporters, which do not contain the DD. Goat polyclonal antibody to tdTomato (red fluorescent protein). RLU = relative light units. FP = fluorescent protein (AmCyan1, ZsGreen1, or tdTomato). We suggest that 3 fluorescent proteins can be easily combined (EGFP or Venus, mTagBFP2 and either mIFP or TagRFP657) without significant compensation or spillover spread (Supplementary Table 1). Cells used to monitor uninduced promoters (e.g., the negative control) will be treated with Shield1 alone. Terms and conditions Can be detected on almost all cytometers. Cells were gated on CD19-/CD56- events, and CD154 (CD40L) PE/Fire 640 is displayed against CD4 PE/Fire 700 or CD8 PerCP. Specifically, five proteins: EGFP, Venus, mTagBFP2, mIFP, and TagRFP657, can be easily utilized (EGFP or Venus, mTagBFP2 and either mIFP or TagRFP657). Provides an intensely bright signal and is ideally used to stain an antigen that is expressed on a small subset of cells. Other fluorescent proteins, like eGFP, mCherry, and tdTomato can be fused to a protein of interest to visualize or track cell processes. tdTomato Fluorescent Protein tdTomato is a bright orange fluorescent protein derived from Discosoma sp. Few, if any, fluors spill into KIRAVIA Blue 520. With each new option, researchers and colleagues have come to trust the performance of our products, as evidenced by this CiteAb report indicating we are the most cited company for flow cytometry antibodies. The https:// ensures that you are connecting to the This very bright red fluorescent protein was made even brighter by creating atandem dimer version, the tdTomato fluorescent protein. It is very useful for adding into a multicolor panel and can be easily analyzed on a bivariate plot versus most other fluors. 293T (Clontech, Mountain View, CA) cells were maintained in Opti-MEM supplemented with L-glutamine and 10% FBS. This causes unwanted spillover into the APC or Alexa Fluor 647 channel and can make population resolution challenging. Nat. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com. The lower brightness on this dye makes it suitable for antigens with high expression levels. These vectors were then packaged into lentivirus using the Lenti-X packaging single shots following the provided protocol. 22, 156772 (2004). 293T cells were transduced with combinations of viral vectors and fluorescence was assayed after 48 hours. To transduce mK4 cells, the DMEM media was removed and replaced with retrovirus-containing media from the 293T cells. Cells were gated on scatter parameters, then single cells were selected using the height and width pulses of FSC and SSC, respectively. Neoplasia 8, 796806 (2006). Note: In our model, tdTomato was driven by the Rosa26 promoter, . Acta Cystallogr. Supplementary Figure 1 - Representative gating scheme. (0)1.3904.6880 Japan: +81. Plots for co-expression (orange) were overlaid with single fluorescent protein expression data (red or blue). 2004). 8,173,792 and 9,487,787. The authors acknowledge support from the Katie Linz Foundation (to D.H.) and a POST award from Alexs Lemonade Stand Foundation (to B. K.) and RO1HL122661 (to H.L.G. Consistent, high-efficiency transfections lead to high titers. We demonstrate the utility of five fluorescent proteins with optimal characteristics for multicolor experiments, three of which can be easily used simultaneously (GFP or Venus, mTagBFP2 and either mIFP or TagRFP657). Can be unmixed from BV421 with more ease than other similar fluorophores on a spectral cytometer. EGFP (MIGR1; AddGene #27490), tdTomato-N1 (Addgene #54642) and pMSCV-mCherry (AddGene #52114) were obtained from AddGene. Can be used with APC and APC/Cyanine7 on conventional flow cytometers. Finally, mTagBFP2, while only moderately bright based on the SI, was easily compatible with the other fluorescent proteins based on its spectral overlap and minimal spillover spreading. Supplementary Table 1 Spillover Spread Matrix of the fluorescent proteins ustilized in the co-expression assay of Fig 2. Compensation was performed using OneComp eBeads (Thermo Fisher Scientific), Anti-Rat Ig, /Negative Control Compensation Particles Set (BD Biosciences), or AbC total antibody compensation bead kit (Thermo Fisher Scientific). 632180), the vector generates high titers of replication-incompetent, VSV-G-pseudotyped lentivirus. Excited off of the 488 nm blue laser and emits between the peak emission of Alexa Fluor 488 or FITC and PE. Properties of lasers and filters used in flow cytometry and confocal microscopy experiments are listed. Not all vector elements are shown. Samples wereunmixed on a 5-laser Cytek Aurora Cytometer using cells. If a marker may be upregulated, it is useful to put it on PE/Cyanine7 because it is the furthest red-shifted fluorophore excited by the 488/561 nm lasers. Antibodies that detect tdTomato can be used in several scientific applications, including Western Blot. Bolded histograms (with small black box) indicate detection channel for the fluorescent protein based on its reported max excitation and emission spectra. Comparable assessments of combinations of fluorescent proteins exist for microscopy, underscoring the need for similar experiments in the context of flow cytometry [54]. 2001. Jewelry. Takara Bio USA, Inc. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. Use our. (B) Alternative emission filters for the detection of EYFP/Venus with EGFP; RFP657 with mIFP; mApple with mCherry; and mCherry with tdTomato. Basic fluorescent protein vector map. Our mission is to develop high-quality innovative tools and services to accelerate discovery. All plots are gated on lymphocytes. official website and that any information you provide is encrypted Transduction of neural progenitor cells by Lenti-X lentivirus. 2023 Takara Bio Inc. All Rights Reserved. Then, the mixture was added to cultured Lenti-X 293T cells that were approximately 80% confluent. tdTomato's emission wavelength (581 nm) and brightness make it ideal for live animal imaging studies. 2004). (Left) Anti-human CD3 (clone UCHT1) BV421 and anti-human T-bet (clone 4B10) KIRAVIA Blue 520 staining on human PBMCs. More photostable than FITC, and thus, more useful in microscopy. To package the vector into high-titer, replication-incompetent lentivirus, we recommend using Lenti-X Packaging Single Shots and the Lenti-X 293T Cell Line. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. In 22 of the 34 mice, a low percentage of hepatocytes were tdTomato + (<13%); in 12 of the 34 mice, we observed high percentages of tdTomato + cells. 0.02% NaN3 and 50% glycerol with final concentration 5mg/mL beads: Conjugation: Magnetic Beads: Storage: Shipped at -20C or with ice packs, Upon delivery store at -20C. To assess spectral overlap and the efficacy of combining certain fluorescent proteins into a single panel, we calculated compensation values and a spillover spreading matrix (SSM) (Fig 2A, Supplementary Table 1, Supplementary Fig 2). Potent tdTomato fluorescence in LSECs was observed for LNPs with 100 nm in size (1.0% PEG-lipid) that is similar to the diameter of fenestrae. Accessibility Generating knock-in gene-expression or fusion-protein reporters is now more tractable because of recent advances in genome engineering. Through our Takara, Clontech, and Cellartis brands, our mission is to develop high-quality innovative tools and services to accelerate discovery. More temperature and photo-stable alternative for APC/Cyanine7. SI is also influenced by instrument sensitivity and PMT voltage as well as auto-fluorescence based on cell type. Sensitivity Index (SI) gives a value for the detection sensitivity of a fluorescent protein using a specific laser and filter set. Instrument setup and QC was performed by core facility staff per manufacturer instructions. Mouse monoclonal tdTomato antibody, Magnetic beads from OriGene Technologies. Lysed human whole blood was stained with the indicated antibodies in the presence of Brilliant Stain Buffer. Academic research institutions are granted an automatic license with the purchase of this product to use the Tet Technology only for internal, academic research purposes, which license specifically excludes the right to sell, or otherwise transfer, the Tet Technology or its component parts to third parties. Best suited for spectral cytometers but can also be used on advanced conventional cytometers that are capable of detecting PE/Cyanine5.5. mTagBFP2 is especially versatile based on the data shown here. These are typically derived from sea anemone and jellyfish, and exist as either monomers, dimers, or trimers. Takara Bio Europe is a member of the Takara Bio Group, a leading life sciences company that is committed to improving the human condition through biotechnology. Shaner, N. C. et al. The pol gene is fused to vpr to ensure transport of the reverse transcriptase/integrase protein into the recombinant lentiviral particle. We note that TagRFP657 has a higher SI (Fig 1) and, therefore, is preferable over mIFP. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). Any use of this product for any of the above mentioned purposes requires a license from the Massachusetts Institute of Technology. Brilliant Violet dyes are organic, light-emitting, diode polymers whose brightness is related to the length of the polymer, but whose spectra remain stable no matter the polymers length. The Mouse Monoclonal tdTomato Antibody, Magnetic Beads has been validated for the following applications: Precipitation, Purification, Protein Purification, and Immunoprecipitation. One of the brightest fluorophores and provides high signal-to-noise ratio. Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Positive cells were collected and expanded. In order to address the need for concise data on the flow cytometric properties of fluorescent proteins that can be used for knock-in gene-expression or fusion-protein reporters, we selected a panel of 13 monomeric fluorescent proteins to examine: Venus [45], monomeric Infrared Fluorescent Protein (mIFP) [41], Long Stokes Shift monomeric Orange (LssmOrange) [46], Tag Red Fluorescent Protein 657 (TagRFP657) [47], monomeric Orange2 (mOrange2) [48], monomeric Apple(mApple) [48], Sapphire [49], monomeric Tag Blue Fluorescent Protein (mTagBFP2) [50], tdTomato [11], monomeric Cherry (mCherry) [11], Enhanced Yellow Fluorescent Protein (EYFP) [51], monomeric Cerulean3 (mCerulean3) [52], and Enhanced Green Fluorescent Protein (EGFP) [53]. Lenti-X Packaging Single Shots (VSV-G) provide an extremely simple and consistent one-step method for producing high-titer lentivirus. yellow fluorescent protein, Improving FRET dynamic range with bright green and red fluorescent proteins, Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins, A far-red emitting fluorescent marker protein, mGarnet2, for microscopy and STED nanoscopy, Extended Stokes shift in fluorescent proteins: chromophoreprotein interactions in a near-infrared TagRFP675 variant, Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine, Acta Crystallographica Section D: Biological Crystallography, Cysteine Sulfoxidation Increases the Photostability of Red Fluorescent Proteins, A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein, A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum, Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging, An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging, Near-infrared fluorescent proteins for multicolor in vivo imaging, A monomeric red fluorescent protein with low cytotoxicity, mBeRFP, an improved large stokes shift red fluorescent protein, Time-resolved flow cytometry for lifetime measurements of near-infrared fluorescent proteins. An MCS at the 3' end of the encoding sequence makes it possible to clone in a gene of interest in order to express it as a C-terminal fusion protein with tdTomato. Effectively replaces PerCP conjugates in an assay, as Cyanine5.5 stabilizes the environmental sensitivity of PerCP. Relatively small, and thus advantageous over APC for intracellular and transcription factor staining. On a spectral unmixing cytometer, PE/Cyanine5 can be distinctly identified from APC and Alexa Fluor 647, allowing for expansion the number of parameters in a multicolor panel. SI gives a value for the detection sensitivity of a fluorescent protein when using a specific laser and filter set. Human PBMCs were incubated with no monocyte blocker (left) or with 5 L of True-Stain Monocyte Blocker (right) and then stained with 5 L/test of the indicated antibodies in 100 L of cells at 1 x 10. Similar spectra to Pacific Orange, but offers a brighter signal overall. Minimal non-specific binding to monocytes. EGFP, mApple and Venus displayed a moderate level of sensitivity compared to the other proteins. The separation of the gag, pol, and env genes effectively reduces the incidence of RCL (Wu et al., 2000). Brilliant Violet dyes are effective over a wide pH range and are compatible with a variety of fixatives. PerCP/Cyanine5.5 may exhibit non-specific binding to monocytes and macrophages. The system provides enough reagents for 16 packaging reactions. Supplementary Figure 2 Specificity of fluorescent protein co-expression. 1Division of Immunobiology and Center for Systems Immunology, Cincinnati Childrens Hospital Medical Center, Cincinnati, Ohio 45229, USA, 2Division of Developmental Biology, Perinatal Institute, Cincinnati Childrens Hospital Medical Center, Cincinnati, OH 45229, 3Division of Rheumatology, Cincinnati Childrens Hospital Medical Center, Cincinnati, Ohio 45229, USA, 4Division of Experimental Hematology and Cancer Biology, Cincinnati Childrens Hospital Medical Center, Cincinnati, Ohio 45229, USA. The Lenti-X DD Red Reporter System includes the Lenti-X DD-tdTomato Vector Set and Shield1. Tandem dye using APC as the donor and Cyanine7 as the acceptor fluorophore. Given its low brightness, it should be paired with antigens with high expression levels.. R-phycoerythrin is composed of phycobilin chromophores embedded in a protein scaffolding. No cross-beam excitation with the red laser. ptdTomato encodes a tandem sequence of the dimeric red fluorescent protein, Tomato. Another protein-based dye, PerCP, a peridinin-chlorophyll protein complex, is derived from photosynthetic dinoflagellates. Control beads were prepared using the AbC Total Antibody Compensation Bead Kit (Invitrogen) and ArC Amine Reactive Compensation Bead Kit (Invitrogen) following manufacturer guidelines. The site is secure. 13. We suggest that three fluorescent proteins can easily be combined and more colors can be selected based on the data shown. In the absence of the membrane-permeant ligand Shield1, the DD causes the reporter protein to be rapidly targeted to and degraded by proteasomes. tdTomato and EGFP also make a good combination due to minimal spillover and spreading error. 54.2 kDa FPbase ID: PGG5S Attributes Ex Em EC(M-1cm-1) Single cells were displayed on all subsequent histograms or dot plots. One of the largest fluorophores and may not be ideal for intracellular targets. tdTomato's emission wavelength (581 nm) and brightness make it ideal for live animal imaging studies. We calculated SI using the following formula: SI = (median fluorescent cells - median non-expressing cells) / (robust SD) where robust SD = (84 percentile non-expressing cells) / (median non-expressing cells) [17]. As such, they may have spillover into equivalent channels on those lasers.