Flow cytometric analysis of RORγt expression in Mouse thymocytes. C57BL/6 Mouse thymocytes were stained with APC Rat Anti-Mouse CD4 (Cat. No. 553051) and BD Horizon™ BUV395 Rat Anti-Mouse CD8a (Cat. No. 563786) antibodies, and then fixed and permeabilized using the BD Pharmingen™ Transcription Factor Buffer Set (Cat. No. 562574). The fixed and permeabilized cells were then stained with either BD Horizon™ RB613 Mouse IgG2a, κ Isotype Control (Cat. No. 571196) or BD Horizon™ RB613 Mouse Anti-Mouse RORγt antibody (Cat. No. 571115/571197) at 0.5 µg/test. The overlapping histograms show the levels of either IgG2a Isotype Control (Left Panel) or RORγt (Right Panel) staining in CD4+CD8- single-positive (dashed line histograms) or CD4+CD8+ double-positive (solid line histograms) 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 FACSymphony™ A5 SE Cell Analyzer System and FlowJo™ Software. Data shown on this Technical Data Sheet are not lot specific.
Flow cytometric analysis of RORγt expression in Mouse thymocytes. C57BL/6 Mouse thymocytes were stained with APC Rat Anti-Mouse CD4 (Cat. No. 553051) and BD Horizon™ BUV395 Rat Anti-Mouse CD8a (Cat. No. 563786) antibodies, and then fixed and permeabilized using the BD Pharmingen™ Transcription Factor Buffer Set (Cat. No. 562574). The fixed and permeabilized cells were then stained with either BD Horizon™ RB613 Mouse IgG2a, κ Isotype Control (Cat. No. 571196) or BD Horizon™ RB613 Mouse Anti-Mouse RORγt antibody (Cat. No. 571115/571197) at 0.5 µg/test. The overlapping histograms show the levels of either IgG2a Isotype Control (Left Panel) or RORγt (Right Panel) staining in CD4+CD8- single-positive (dashed line histograms) or CD4+CD8+ double-positive (solid line histograms) 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 FACSymphony™ A5 SE Cell Analyzer System and FlowJo™ Software. Data shown on this Technical Data Sheet are not lot specific.
Product Details
BD Horizon™
RORγT; RORgt; RORgamma t; RORgammaT; Rorc2; Rorg; TOR; Thor; Nr1f3
Mouse (QC Testing)
Mouse IgG2a, κ
Mouse RORγt Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
19885
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO
Preparation And Storage
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to the dye under optimum conditions and unreacted dye was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
Recommended Assay Procedures
BD® CompBeads can be used as surrogates to assess fluorescence spillover (compensation). When fluorochrome conjugated antibodies are bound to BD® 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 BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.
Product Notices
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- When using high concentrations of antibody, background binding of this dye to erythroid fragments produced by ammonium chloride-based lysis, such as with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899), has been observed when the antibody conjugate was present during the lysis procedure. This may cause nonspecific staining of target cells, such as leukocytes, which have bound the resulting erythroid fragments. This background can be mitigated by any of the following: titrating the antibody conjugate to a lower concentration, fixing samples with formaldehyde, or removing erythrocytes before staining (eg, gradient centrifugation or pre-lysis with wash). This background has not been observed when cells were lysed with BD FACS™ Lysing Solution (Cat. No. 349202) after staining.
- An isotype control should be used at the same concentration as the antibody of interest.
- Please observe the following precautions: We recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to protect exposure of conjugated reagents, including cells stained with those reagents, to any room illumination. Absorption of visible light can significantly affect the emission spectra and quantum yield of tandem fluorochrome conjugates.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- CF™ is a trademark of Biotium, Inc.
- For U.S. patents that may apply, see bd.com/patents.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
RB613 Mouse IgG2a, κ Isotype Control RUO
Size
0.1 mg
Cat No.
571196
Transcription Factor Buffer Set RUO
Size
100 Tests
Cat No.
562574
APC Rat Anti-Mouse CD4 RUO
Size
0.1 mg
Cat No.
553051
BUV395 Rat Anti-Mouse CD8a RUO
Size
50 µg
Cat No.
563786
571115 Rev. 1
Antibody Details
Q31-378
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.
571115 Rev. 1
Format Details
RB613
The BD Horizon RealBlue™ 613 (RB613) Dye is part of the BD® family of blue dyes. It is a tandem fluorochrome with an excitation maximum (Ex Max) at 492-nm and an emission maximum (Em Max) at 613-nm as measured using an antibody-dye conjugate. Driven by BD® innovation, RB613 can be used on both spectral and conventional cytometers and is designed to be excited by the Blue laser (488-nm) with reduced excitation by the 561-nm Yellow-Green laser. For conventional instruments equipped with a Blue laser (488-nm), RB613 can be used as an alternative to PE-CF594 and we recommend using an optical filter centered near 610-nm (eg, a 610/20-nm bandpass filter). For spectral instruments equipped with a Blue laser (488-nm), it can be used in conjunction with PE-CF594. RB613 is on average brighter than PE-CF594 off the blue laser.
RB613
492 nm
613 nm
571115 Rev.1
Citations & References
View product citations for antibody "571115" on CiteAb
Development References (10)
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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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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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Lee JY, Hall JA, Kroehling L, et al. Serum Amyloid A Proteins Induce Pathogenic Th17 Cells and Promote Inflammatory Disease. Cell. 2020; 180(1):79-91.e16. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
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Metidji A, Omenetti S, Crotta S, et al. The Environmental Sensor AHR Protects from Inflammatory Damage by Maintaining Intestinal Stem Cell Homeostasis and Barrier Integrity.. Immunity. 2018; 49(2):353-362.e5. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
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Sidwell T, Liao Y, Garnham AL, et al. Attenuation of TCR-induced transcription by Bach2 controls regulatory T cell differentiation and homeostasis. Nature communications. 2020; 11(1):252. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
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Wang H, Hogquist KA. CCR7 defines a precursor for murine iNKT cells in thymus and periphery. eLife. 2018; 7:e34793. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
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Wei SC, Sharma R, Anang NAS, et al. Negative Co-stimulation Constrains T Cell Differentiation by Imposing Boundaries on Possible Cell States. Immunity. 2019; 50(4):1084-1098.e1010. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
571115 Rev. 1
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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.