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Multicolor flow cytometric analysis of RORγt expression in mouse thymocytes. BALB/c mouse thymocytes were stained with BD Horizon™ BUV395 Rat Anti-Mouse CD4 (Cat. No. 740208) and APC Rat Anti-Mouse CD8a (Cat. No. 553035/561093) antibodies, fixed and then permeabilized using the BD Pharmingen™ Transcription Factor Buffer Set (Cat. No. 562574/562725). The fixed and permeabilized cells were then stained with BD Horizon™ BB515 Mouse Anti-Mouse RORγt antibody (Cat. No. 567175) at 0.125 µg/test. The overlapping histograms show the levels of RORγt expression in CD4+ CD8- single-positive (dashed line histogram) or CD4+ CD8+ double-positive (solid line histogram) thymocytes. The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact cells. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
BD Horizon™ BB515 Mouse Anti-Mouse RORγt
Regulatory Status Legend
Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
Preparation And Storage
Recommended Assay Procedures
BD™ CompBeads can be used as surrogates to assess fluorescence spillover (Compensation). When fluorochrome conjugated antibodies are bound to CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and CompBead to ensure that BD Comp beads are appropriate for your specific cellular application.
For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime two or more BD Horizon Brilliant dyes are used in the same experiment. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. No. 566385).
For optimal results, it is recommended to perform 2 washes after staining with antibodies. Cells may be prepared, stained with antibodies and washed twice with wash buffer per established protocols for immunofluorescence staining, prior to acquisition on a flow cytometer. Performing fewer than the recommended wash steps may lead to increased spread of the negative population.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- BD Horizon Brilliant Stain Buffer is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,575,303; 8,354,239.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- Alexa Fluor™ is a trademark of Life Technologies Corporation.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
The Q31-378 monoclonal antibody recognizes mouse RORgamma t (RORγt), an isoform of RORgamma (RORγ). RORγt is a DNA-binding transcription factor that belongs to the ROR/RZR orphan nuclear receptor family. RORγt is expressed exclusively by lymphoid cells including CD4+CD8+ thymocytes, peripheral CD4+ Th17 and CD8+ Tc17 cells, NKT cells and innate lymphoid cells such as lymphoid tissue inducer (LTi) cells. RORγt plays essential roles in thymopoiesis, T cell homeostasis, differentiation of effector T lymphocytes and the development of secondary lymphoid tissues including lymph nodes and Peyer's patches.
The antibody was conjugated to BD Horizon BB515 which is part of the BD Horizon Brilliant™ Blue family of dyes. With an Ex Max near 490 nm and an Em Max near 515 nm, BD Horizon BB515 can be excited by the blue laser (488 nm) laser and detected with a 530/30 nm filter. This dye has been exclusively developed by BD Biosciences and is up to seven times brighter than FITC with less spillover into the PE channel. Due to similar excitation and emission properties, BB515, FITC, and Alexa Fluor™ 488 cannot be used simultaneously. It is not recommended to use BB515 in cocktails that include Streptavidin conjugates as it may cause high background.
Development References (10)
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Bechtel S, Rosenfelder H, Duda A, et al. The full-ORF clone resource of the German cDNA Consortium. BMC Biochem. 2007; 8:399-410. (Biology). View Reference
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Eberl G, Littman DR. The role of the nuclear hormone receptor RORgt in the development of lymph nodes and Peyer's patches. Immunol Rev. 2003; 195(195):81-90. (Biology). View Reference
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Eberl G, Marmon S, Sunshine MJ, Rennert PD, Choi Y, Littman DR. An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells. Nat Immunol. 2004; 5(1):64-73. (Biology). View Reference
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Hamada H, Garcia-Hernandez MdlL, Reome JB, et al. Tc17, a unique subset of CD8 T cells that can protect against lethal influenza challenge. J Immunol. 2009; 182(6):3469-3481. (Biology). View Reference
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He YW, Deftos ML, Ojala EW, Bevan MJ. RORgamma t, a novel isoform of an orphan receptor, negatively regulates Fas ligand expression and IL-2 production in T cells. Immunity. 1998; 9(6):797-806. (Biology). View Reference
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Hirose T, Smith RJ, Jetten AM. ROR gamma: the third member of ROR/RZR orphan receptor subfamily that is highly expressed in skeletal muscle. Biochim Biophys Acta. 1994; 205(3):1976-1983. (Biology). View Reference
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Ivanov, II, McKenzie BS, Zhou L, et al. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell. 2006; 126(6):1121-1133. (Biology). View Reference
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Medvedev A, Yan ZH, Hirose T, Giguere V, Jetten AM. Cloning of a cDNA encoding the murine orphan receptor RZR/ROR gamma and characterization of its response element. Gene. 1996; 181(1-2):199-206. (Biology). View Reference
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Unutmaz D. RORC2: the master of human Th17 cell programming. Eur J Immunol. 2009; 39(6):1452-1455. (Biology). View Reference
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van de Pavert SA, Ferreira M, Domingues RG, et al. Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity. Nature. 2014; 508(7494):123-127. (Clone-specific: Flow cytometry). View Reference
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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product as described.
Comparisons, where applicable, are made against older BD technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
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