Antibody data
- Antibody Data
- Antigen structure
- References [8]
- Comments [0]
- Validations
- Flow cytometry [1]
- Other assay [4]
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- Product number
- 46-9852-82 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- IRF8 Monoclonal Antibody (V3GYWCH), PerCP-eFluor™ 710, eBioscience™
- Antibody type
- Monoclonal
- Antigen
- Other
- Description
- Description: The V3GYWCH monoclonal antibody reacts with human and mouse interferon regulatory factor 8 (IRF8; ICSBP). IRF8 is a 50 kDa transcription factor that plays a critical role in the development of dendritic cells (DC). Specifically, IRF8 is essential in the development and differentiation of plasmacytoid DC (pDC) and CD8a+ DC. IRF8-/- mice are deficient in both pDC and CD8a+ DC populations. CD8a- DC are present in normal numbers in IRF8-/- mice but fail to mature upon Toll-like receptor signaling and are functionally impaired in response to in vivo microbial stimulation. IRF8 deficiency does not affect the frequency or viability of DC precursors and as a result retroviral IRF8 transduction restores pDC development from DC progenitors in IRF8-/- mice. Applications Reported: This V3GYWCH antibody has been reported for use in intracellular staining followed by flow cytometric analysis. Applications Tested: This V3GYWCH antibody has been tested by intracellular staining and flow cytometric analysis of normal human peripheral blood cells using the Foxp3/Transcription Factor Staining Buffer Set (Product # 00-5523-00) and protocol. Please refer to Best Protocols: Protocol B: One-step protocol: intracellular (nuclear) proteins located under the Resources Tab online. This can be used at less than or equal to 0.5 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. PerCP-eFluor® 710 emits at 710 nm and is excited with the blue laser (488 nm); it can be used in place of PerCP-Cyanine5.5. We recommend using a 710/50 bandpass filter, however, the 695/40 bandpass filter is an acceptable alternative. Please make sure that your instrument is capable of detecting this fluorochrome. Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-822-49) (100 µL cell sample + 100 µL IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333-54) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically. Excitation: 488 nm; Emission: 710 nm; Laser: Blue Laser. Filtration: 0.2 µm post-manufacturing filtered.
- Reactivity
- Human, Mouse
- Host
- Mouse
- Isotype
- IgG
- Antibody clone number
- V3GYWCH
- Vial size
- 100 µg
- Concentration
- 0.2 mg/mL
- Storage
- 4° C, store in dark, DO NOT FREEZE!
Submitted references Protein Tyrosine Phosphatase Non-Receptor Type 2 Function in Dendritic Cells Is Crucial to Maintain Tissue Tolerance.
Inflammatory Type 2 cDCs Acquire Features of cDC1s and Macrophages to Orchestrate Immunity to Respiratory Virus Infection.
CD103(+) cDC1 and endogenous CD8(+) T cells are necessary for improved CD40L-overexpressing CAR T cell antitumor function.
Biallelic mutations in IRF8 impair human NK cell maturation and function.
Batf3 selectively determines acquisition of CD8(+) dendritic cell phenotype and function.
Cell-intrinsic expression of TLR9 in autoreactive B cells constrains BCR/TLR7-dependent responses.
Batf3 maintains autoactivation of Irf8 for commitment of a CD8α(+) conventional DC clonogenic progenitor.
Beta-catenin signaling drives differentiation and proinflammatory function of IRF8-dependent dendritic cells.
Hering L, Katkeviciute E, Schwarzfischer M, Busenhart P, Gottier C, Mrdjen D, Komuczki J, Wawrzyniak M, Lang S, Atrott K, Becher B, Rogler G, Scharl M, Spalinger MR
Frontiers in immunology 2020;11:1856
Frontiers in immunology 2020;11:1856
Inflammatory Type 2 cDCs Acquire Features of cDC1s and Macrophages to Orchestrate Immunity to Respiratory Virus Infection.
Bosteels C, Neyt K, Vanheerswynghels M, van Helden MJ, Sichien D, Debeuf N, De Prijck S, Bosteels V, Vandamme N, Martens L, Saeys Y, Louagie E, Lesage M, Williams DL, Tang SC, Mayer JU, Ronchese F, Scott CL, Hammad H, Guilliams M, Lambrecht BN
Immunity 2020 Jun 16;52(6):1039-1056.e9
Immunity 2020 Jun 16;52(6):1039-1056.e9
CD103(+) cDC1 and endogenous CD8(+) T cells are necessary for improved CD40L-overexpressing CAR T cell antitumor function.
Kuhn NF, Lopez AV, Li X, Cai W, Daniyan AF, Brentjens RJ
Nature communications 2020 Dec 2;11(1):6171
Nature communications 2020 Dec 2;11(1):6171
Biallelic mutations in IRF8 impair human NK cell maturation and function.
Mace EM, Bigley V, Gunesch JT, Chinn IK, Angelo LS, Care MA, Maisuria S, Keller MD, Togi S, Watkin LB, LaRosa DF, Jhangiani SN, Muzny DM, Stray-Pedersen A, Coban Akdemir Z, Smith JB, Hernández-Sanabria M, Le DT, Hogg GD, Cao TN, Freud AG, Szymanski EP, Savic S, Collin M, Cant AJ, Gibbs RA, Holland SM, Caligiuri MA, Ozato K, Paust S, Doody GM, Lupski JR, Orange JS
The Journal of clinical investigation 2017 Jan 3;127(1):306-320
The Journal of clinical investigation 2017 Jan 3;127(1):306-320
Batf3 selectively determines acquisition of CD8(+) dendritic cell phenotype and function.
Chandra J, Kuo PT, Hahn AM, Belz GT, Frazer IH
Immunology and cell biology 2017 Feb;95(2):215-223
Immunology and cell biology 2017 Feb;95(2):215-223
Cell-intrinsic expression of TLR9 in autoreactive B cells constrains BCR/TLR7-dependent responses.
Nündel K, Green NM, Shaffer AL, Moody KL, Busto P, Eilat D, Miyake K, Oropallo MA, Cancro MP, Marshak-Rothstein A
Journal of immunology (Baltimore, Md. : 1950) 2015 Mar 15;194(6):2504-12
Journal of immunology (Baltimore, Md. : 1950) 2015 Mar 15;194(6):2504-12
Batf3 maintains autoactivation of Irf8 for commitment of a CD8α(+) conventional DC clonogenic progenitor.
Grajales-Reyes GE, Iwata A, Albring J, Wu X, Tussiwand R, Kc W, Kretzer NM, Briseño CG, Durai V, Bagadia P, Haldar M, Schönheit J, Rosenbauer F, Murphy TL, Murphy KM
Nature immunology 2015 Jul;16(7):708-17
Nature immunology 2015 Jul;16(7):708-17
Beta-catenin signaling drives differentiation and proinflammatory function of IRF8-dependent dendritic cells.
Cohen SB, Smith NL, McDougal C, Pepper M, Shah S, Yap GS, Acha-Orbea H, Jiang A, Clausen BE, Rudd BD, Denkers EY
Journal of immunology (Baltimore, Md. : 1950) 2015 Jan 1;194(1):210-22
Journal of immunology (Baltimore, Md. : 1950) 2015 Jan 1;194(1):210-22
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
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- Experimental details
- Staining of normal human peripheral blood cells with Anti-Human CD123 PE (Product # 12-1239-42) followed by fixation and permeablization using the Foxp3/Transcription Factor Staining Buffer Set (Product # 00-5523-00) and protocol. Cells were then stained intracellularly with 0.25 µg of Mouse IgG1 K Isotype Control PerCP-eFluor® 710 (Product # 46-4714-82) (left) or 0.25 µg of Anti-Human/Mouse IRF8 PerCP-eFluor® 710 (right). Total viable cells, as determined by Fixable Viability Dye eFluor® 450 (Product # 65-0863-14), were used for analysis.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- NULL
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- NULL
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Figure 4 IRF4-expressing CD8+ DCs in Batf3-/- mice are able to process antigen but lose capacity to take up dying cells (a) Splenocytes of Batf3-/- and control mice were incubated with DQ-OVA for 3 hours and subsequently analysed by flow cytometry for DQ-OVA processing. CD8+ and CD11b+ dendritic cell subsets were gated as shown in Figure 2 . Shown are representative histograms of green fluorescence (DQ-OVA) of treated versus untreated CD8+ and CD11b+ DCs in Batf3-/- and control splenocytes. The percentage and MFI of DQ-OVA+ CD8+ or CD11b+ DCs as well as of untreated samples (-) was compared. Each point represents a sample of one animal with indication of mean +/- SD of whole group (n=4). Shown is one of two independent experiments. (b) Splenocytes of Batf3-/- and control mice were incubated with DiI-labelled liposomes for 90 min and subsequently analysed by flow cytometry for liposome uptake. Shown are representative histograms of red fluorescence (DiI+) of CD8+ and CD11b+ DCs in Batf3-/- and control splenocytes incubated at 37degC or on ice. The percentage of DiI+ cells of CD8+ or CD11b+ DCs was compared. Each point represents a sample of one animal with indication of mean+/- SD of the whole group (n=5). (c-d) CellTrace Violet labelled, UV irradiated dying cells were injected into Batf3-/- and control mice. 3 hours later, splenic CD11b+ and CD8+ DCs were analysed for endocytosis of dying cells. (c) Shown are flow cytometry plots that were pre-gated on DCs as shown in Supplem
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Fig. 3 m1928z-CD40L CAR T cells stimulate tumor-resident CD11b-CD103- DN cDCs to proliferate, upregulate IRF8, and differentiate to cDC1s. a IRF8 expression in CD11b - CD103 - double-negative (DN) (orange), CD11b - CD103 + cDC1 (green), and CD11b + CD103 + cDC2 (blue) in the tumor of untreated A20.GL tumor-bearing mice. FMO, flow minus one. b - d Ki-67 and IRF8 expression shown as flow cytometry contour plots in CD11b - CD103 - DN cDCs in the tumor ( b ), spleen ( c ), and tumor-draining lymph nodes (tdLN) ( d ) of A20.GL tumor-bearing mice on day 7 after CAR T cell treatment. Percentage of Ki-67 + IRF8 + DN cDCs is summarized from two independent experiments ( n = 7/group). e CD45.2 + A20.GL tumor-bearing mice were treated with 3 x 10 6 CAR T cells i.v. and CD45.2 + CD11b - CD103 - DN cDCs were isolated from the tumor on day 3 by FACS. Sorted CD45.2 + DN cells were cultured in vitro on a CD45.1 + bone-marrow stromal layer for 3 days and the percentage of CD11c + CD103 + cDC1s of all CD45.2 + cells was analyzed. Shown are representative contour plots and the quantification of the percentage of CD11c + CD103 + cDC1s. Each dot represents one in vitro culture. Data were collected from two independently performed experiments (m1928z, n = 5; m1928z-CD40L, n = 6). f , g Ki-67 expression shown as contour plots in CD11b - CD103 + cDC1s ( f ) and CD11b + CD103 - cDC2s ( g ) in the tumor of A20.GL tumor-bearing mice on day 7 after CAR T cell treatment. Percentage of Ki-67 + cells is su