17-9852-80

antibody from Invitrogen Antibodies

Targeting: IRF8 ICSBP, ICSBP1, IRF-8

Provider product page for 17-9852-80

Flow cytometry

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Antibody data

Antibody Data
Antigen structure
References [14]
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Validations
Flow cytometry [1]
Other assay [7]

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Product number: 17-9852-80 - Provider product page
Provider: Invitrogen Antibodies
Product name: IRF8 Monoclonal Antibody (V3GYWCH), APC, 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 0.25 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. Excitation: 633-647 nm; Emission: 660 nm; Laser: Red Laser. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human, Mouse
Host: Mouse
Isotype: IgG
Antibody clone number: V3GYWCH
Vial size: 25 µg
Concentration: 0.2 mg/mL
Storage: 4° C, store in dark, DO NOT FREEZE!

IRF8 protein structure - 17-9852-80 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Activated inf-cDC2s Share Characteristics with cDC1 and MC Subsets (A and B) Proliferation profile of CTV-labeled CD4 + (A) and CD8 + (B) PVM-specific TCR transgenic T cells cocultured for 4 days with different migratory cDC subsets sorted from 4-6 pooled MLNs 8 dpi with PVM (see also Figures S1 K-S1P). (C) Number of divided CD4 + and CD8 + PVM TCR transgenic T cells cocultured for 4 days with different migratory cDC subsets sorted from MLNs 8 dpi with PVM relative to the cDC2 subset, which is set to 0. Error bars represent 95% confidence intervals. Data were pooled from 3 independent experiments (n = 5-7). (D) IFN-gamma measured in supernatants of cocultured CD4 + and CD8 + PVM TCR transgenic T cells for 4 days with different MLN-derived cDC subsets sorted 8 dpi with PVM. Data are representative of 3 independent experiments. Error bars indicate +- SEM. Mann-Whitney U test; * p < 0.05, ** p < 0.01. (E) Proliferation profile of CTV-labeled CD4 + and CD8 + PVM TCR transgenic T cells cocultured for 4 days with T cells alone, inf-cDC2s, or MCs sorted from 3 pooled lungs 8 dpi with PVM. For peptide controls, 1 mug/mL of CD4 + (M37-M47) or CD8 + (N339-N347) immunodominant epitope was added ex vivo . (F-H) Percentage of CD86-expressing (F) and CCR7-expressing (G) and IL-12-producing (H) lung or migratory DC subsets in MLNs upon mock infection (white circles) or 10 dpi with PVM (black dots). For IL-12 staining, samples were restimulated ex vivo for 6 h. Data are representati

Supportive validation

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Experimental details: Figure 3 IRF8 Controls the Gene Network in inf-cDC2s (A) Expression of IRF4 (top panel) and IRF8 (bottom panel) by different DC subsets in the lungs and MLNs 10 dpi with PVM. (B) Normalized Median Fluoresent Intensity (MFI) for IRF4 and IRF8 relative to the cDC2 subset, which is set to 0 for DC subsets in the lungs (top panel) or the different migratory cDC subsets in MLNs (bottom panel) 10 dpi with PVM. Error bars represent 95% confidence intervals. Data are representative of 3 independent experiments with 4-6 mice per group. (C) Schematic representation of CD45.1 WT:CD45.2 Irf8 fl/fl Itgax - cre BM chimeras. (D) Normalized CD45.1/CD45.2 ratio relative to B cells of DC subsets in the lungs (left panel) and migratory cDCs in MLNs (right) 10 dpi with PVM. Data are representative of 2 independent experiments with 4-6 mice per group, analyzed with a one-way ANOVA with Sidak correction for multiple comparisons. Error bars indicate +- SEM. * p < 0.05, ** p < 0.01; ns, not statistically significant. (E) UMAP plot of scRNA-seq data of pooled, sorted, live CD3-CD19-SiglecF-CD11c + MHCII + cells from lungs of CD45.1 WT:CD45.2 Irf8 fl/fl Itgax - cre chimeric mice 10 dpi with PVM, showing assigned clusters, and UMAPs showing expression of key annotation markers by color (gray, low expression; blue, high expression) (see also Figures S5 A-S5C). (F) UMAP plot overlay (left panel) similar to (E) but with the colors representing origin of the WT (teal) or Irf8 fl/fl Itgax -cre (red) compart

Supportive validation

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Experimental details: Figure 1 IRF-8 expression on monocytes and dendritic cells. (A) Intracellular IRF-8 levels, expressed as geometric mean fluorescence intensity (GMF), on monocyte subsets (classical, intermediate, and non-classical) and dendritic cell (DC) subsets (myeloid and plasmacytoid). The hash mark indicates the median and error bars are the interquartile range. ** p < 0.01; *** p < 0.001, **** p < 0.0001. (B) Representative histograms of IRF-8 expression in each subset.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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