46-9858-82

antibody from Invitrogen Antibodies

Targeting: IRF4 LSIRF, MUM1

Provider product page for 46-9858-82

Flow cytometry

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Antibody data

Antibody Data
Antigen structure
References [10]
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Validations
Flow cytometry [1]
Other assay [11]

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Product number: 46-9858-82 - Provider product page
Provider: Invitrogen Antibodies
Product name: IRF4 Monoclonal Antibody (3E4), PerCP-eFluor™ 710, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The monoclonal antibody 3E4 reacts with human and mouse interferon regulatory factor 4 (IRF4). IRF4 is a 52 kDa transcription factor with roles in B cell, T cell and macrophage function. In B cells, IRF4 is highly expressed in mature plasma cells and plays a crucial role in their differentiation. IRF4 has been shown to interact with PU.1 and control the transcription of many B cell-specific genes including Prdm1, which encodes Blimp1. In T cells, IRF4 has been implicated in regulatory T (Treg), Th2, Th9 and Th17 cell development and function. This transcription factor is upregulated upon T cell activation and is expressed in mature T cells. Studies have shown that IRF4 directly induces Blimp1 expression in Tregs, leading to IL-10 expression. IRF4 has also been demonstrated to be involved in macrophage polarization and regulation. Lastly, in addition to its roles in normal immune function, IRF4 expression has been reported to be upregulated in many blood-related cancers. Applications Reported: This 3E4 antibody has been reported for use in intracellular staining followed by flow cytometric analysis. Applications Tested: This 3E4 antibody has been tested by intracellular staining and flow cytometric analysis of stimulated normal human peripheral blood cells using the Foxp3/Transcription Factor Buffer Set (Product # 00-5523-00) and protocol. Please see Best Protocols Section (Staining intracellular Antigens for Flow Cytometry) for staining protocol (refer to Protocol B: One-step protocol for intracellular (nuclear) proteins. 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: Rat
Isotype: IgG
Antibody clone number: 3E4
Vial size: 100 µg
Concentration: 0.2 mg/mL
Storage: 4° C, store in dark, DO NOT FREEZE!

IRF4 protein structure - 46-9858-82 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)
Main image
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: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4. B cells promote CD8 T cell memory programming following subunit vaccination (A) Representative histograms for Eomes, CD122, IRF4, and TCF1 staining of tetramer+ cells from WT (black) or MD4 (maroon) mice 7 days after combined-adjuvant vaccination or for CD44- naive control CD8 T cells (gray). (B) The gMFI of tetramer+ cells for Eomes, CD122, and IRF4, where the x axis intersects the y axis at the average gMFI value for CD44- naive controls, and the percentage of tetramer+ cells that stained positive for TCF1. (C and D) Representative KLRG1 versus CD127 staining (C) and numbers of tetramer+ splenocytes in the respective quadrants (D), 7 days after immunization. Data shown are means +- SEM; n : mice per group, representative of 3 experiments. Significance was defined by two-tailed, unpaired Student's t tests, where *p < 0.05, **p < 0.01, and ***p < 0.001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4. ATP-Linked OXPHOS Controls BATF Induction by Regulating TCR Signaling (A and B) Naive CD4 T cells were differentiated under Th1 or Th2 polarizing conditions in the presence of vehicle or oligomycin for 24 h, and expression of (A) BATF, IRF4, and (B) GATA3 was determined by flow cytometry (4 to 5 independent experiments). (C and D) Naive CD4 T cells from Nur77/GFP spleens were activated under Th17 conditions in the presence of vehicle or oligomycin for indicated period of time. (C) Graph shows the expression of Nur77/GFP. (D) Representative plots are gated on live CD4 T cells (representative of 3 independent experiments). (E) Naive CD4 T cells were activated under Th17 conditions with vehicle or oligomycin. Representative histogram overlay shows the phosphorylation of ZAP70 at 24 h (representative of 5 independent experiments). (F) Naive CD4 T cells from Nur77/GFP reporter spleens were stimulated with Th17 cytokines for 6 h. BATF expression in GFP + and GFP - cells were determined by flow cytometry (3 independent experiments). Graphs show the average +- SD; (A and C) two-way ANOVA; (B, E and F) unpaired t test *p < 0.05, **p < 0.01, ***p < 0.001.

Supportive validation

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Experimental details: Figure 2. RNA-Sequencing reveals graded expression of activation signature genes. ( A ) Principal component analysis (PCA) of the approximately 3,200 genes that changed between any two samples reveals that the primary axis of variation (PC1, shown along the x-axis) orders the five conditions by increasing TCR signal strength: No Peptide; low-dose, low-affinity (10 muM K99A); high-dose, low-affinity (100 muM K99A); low-dose, high-affinity (0.1 muM PCC); and high-dose, high-affinity (10 muM PCC). ( B ) After ordering the ~3200 genes used for PCA by their contribution to PC1, we extracted the top 10%--that is, the ~320 genes contributing most positively to a sample's PC1 value--and the bottom 10%--that is, the ~320 genes contributing most negatively to a sample's PC1 value. Each group displays a clear trend, with the top 10% increasing in expression as signal strength increases, and the bottom 10% decreasing in expression. Each blue line represents a gene, with reads per kilobase per million (RPKM) normalized from 0 to 1 across the five conditions. Significance was determined using permutation testing, where the mean difference between genes in the No Peptide sample as compared to 10 muM PCC was normally distributed over randomly generated groups of genes. This normal distribution was compared to the top 10% and bottom 10% genes to generate a p-value. ( C ) Genes in the top 10% of PC1, termed activation signature genes, include many genes previously identified as impor

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 3 Targeted ROCK2 inhibition decreased the binding of ROCK2 to the Irf4 and Bcl6 promoters at Th17-skewing conditions. Human peripheral blood CD4 + T cells were stimulated under Th17-skewing conditions for 48 hours in the absence or presence of KD025, chromatin was purified and proceeded to ChIP-qPCR analyses with anti-ROCK2 or anti-STAT3 antibodies ( a , b ), or Cells were stained with antibodies to IRF4 and analyzed by Flow Cytometry ( c , d ). ( c ) IRF4 expression measured by flow cytometry. Left, Mean Fluorescence Index (MFI) for IRF4 is plotted as percentage of MFI for skewed cells (as 100%). Right, Histogram of the flow cytometry analysis. The average of four different experiments is shown. *p < 0.05; **p < 0.01.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2 Decreased number of plasma cells in Ufbp1 F/F CD19 cre mice. a Bone marrow cells from indicated mice were stained with BLIMP1, CD138, IRF4, and Pax5, and analyzed by flow cytometry. Shown is the BLIMP1 versus CD138 (upper panel), and IRF4 versus Pax5 staining (lower panel) b Enumeration of frequencies of BLIMP1 + CD138 + and IRF4 + Pax5 - cells in a ( n = 3 mice/genotype). c Ufbp1 F/F and Ufbp1 F/F CD19 cre mice immunized with 4-hydroxy-3-nitrophenylacetyl conjugated to keyhole limpet hemocyanin (NP-KLH) as in Fig. 1c were analyzed for plasma cells (BLIMP1 + CD138 + and IRF4 + Pax5 - ) in bone marrow by flow cytometry at the indicated time points after immunization. d Enumeration of the frequencies of plasma cells (BLIMP1 + CD138 + and IRF4 + Pax5 - ) in c ( n = 3 mice/genotype). e Indicated mice were immunized as in Fig. 1c . Twenty-one days later, bone marrow cells were analyzed for the presence of NP-specific IgG1 + ASCs by ELISpot. Shown is a photograph of representative ELISpot wells. f Number (mean +- SEM) of NP-specific IgG1 + ELISpots in e ( n = 6 mice/genotype). g Ufbp1 F/F and Ufbp1 F/F CD19 cre mice were immunized with NP-KLH in Sigma Adjuvant System and on day 10, 7-AAD - CD4 - CD8 - Gr-1 - CD11b - splenic cells were analyzed for IgD and NP staining by flow cytometry. h Enumeration of the frequency of IgD - NP + cells in g ( n = 3 mice/genotype). i BLIMP1 and CD138 expression by IgD - NP + cells in g was an