Antibodies and dilutions used for staining included CD4 (RM4-5, 1:200), CD44 (IM7, 1:200), ICOS (C398.4A, 1:100), TCRβ (H57-597, 1:100), F4/80 (BM8, 1:400), and T-bet (c4B10, 1:50) all from eBioscience; PSGL-1 (2PH1, 1:1000), B220 (RA3-6B2, 1:400), GL-7 (GL7, 1:400), CD95 (Jo2, 1:400), CD138 (281-2, 1:400), CD11b (M1/70, 1:400), Gr-1 (RB6-8C5, 1:400), CD8 (53-6.7, 1:200), Bcl6 (K112-91, 1:50), and CD40L (MR1, 1:50), all from BD Bioscience; PD-1 (RMP1-30, 1:200), IgD (11-26, 1:400), CXCR5 (L138D7, 1:1000), Ki-67 (16A8, 1:100), and IFN-γ (XMG1.2, 1:100), all from BioLegend; granzyme B (GB11, 1:200, Invitrogen) and IL-21R–FC (1:50, R&D Systems). For intracellular cytokine staining, cells were stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) for 2 hours with Golgi Plug (BD Bioscience) for an additional 2 hours. Cells were fixed (BD CytoFix/CytoPerm™) and permeabilized (BD PERM/Wash™) following the manufacturer's protocol. IL-21 was detected by PE-conjugated F(ab')2 (1:400, Jackson ImmunoResearch). For intracellular transcription factor staining, surface molecule-stained cells were fixed and permeabilized using the buffer (eBioscience) without stimulation, and followed by staining for transcription factors. Stained cells were analyzed using a LSRII multilaser flow cytometry (BD) and followed by FlowJo software (Tree Star).
Bcl 6
Manufactured by BD
Bcl-6 is a laboratory equipment product used for the detection and quantification of the Bcl-6 protein. Bcl-6 is a transcriptional repressor that plays a crucial role in the regulation of gene expression related to B-cell development and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by GenScript (4 mentions)
Cd138, by BD (2 mentions)
Ifn γ, by BioLegend (2 mentions)
T bet, by Thermo Fisher Scientific (2 mentions)
Facscanto 2, by BD (2 mentions)
Foxp3, by Thermo Fisher Scientific (2 mentions)
Blimp 1, by GenScript (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Perm wash, by BD (1 mentions)
Il 21r fc, by R&D Systems (1 mentions)
Cd40l, by BD (1 mentions)
Cxcr5, by BioLegend (1 mentions)
Granzyme b, by Thermo Fisher Scientific (1 mentions)
F4 80, by Thermo Fisher Scientific (1 mentions)
Cd11b, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Foxp3, by BD (1 mentions)
Anti igd, by Thermo Fisher Scientific (1 mentions)
Anti pd 1, by Thermo Fisher Scientific (1 mentions)
12 protocols using bcl 6
1
Multiparametric Flow Cytometry Assay
2
Multiparametric Immune Cell Profiling
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Specific cell-surface staining was performed using a standard procedure with anti-CD4, anti-PD1, anti-IgD, anti-GL7 (eBioscience), and anti-CXCR5 mAbs (BD Biosciences).
Intracellular c-Maf, GATA-3, FoxP3, BCL6, Ki67 (Ab from BD Biosciences), and T-bet (eBioscience), staining was performed according to the manufacturer’s protocol (FoxP3 staining set protocol, eBioscience). Cells were analyzed by flow cytometry with a FACS Canto II (BD Biosciences) and analyzed with the FlowJo Software. Live cells were analyzed within a FSC-A/FSC-H gate to exclude cell doublets and triplets.
Intracellular c-Maf, GATA-3, FoxP3, BCL6, Ki67 (Ab from BD Biosciences), and T-bet (eBioscience), staining was performed according to the manufacturer’s protocol (FoxP3 staining set protocol, eBioscience). Cells were analyzed by flow cytometry with a FACS Canto II (BD Biosciences) and analyzed with the FlowJo Software. Live cells were analyzed within a FSC-A/FSC-H gate to exclude cell doublets and triplets.
3
Flow Cytometry Analysis of Lymphocytes
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Lymphocytes of the vaccine-dLNs and spleens were harvested by mashing the tissues through cell strainer (BD Falcon). Antibodies for flow cytometry analysis in the study include CD4 (Biolegend, clone RM4-5), CD45.1 (Biolegend, clone A20), PD-1 (Biolegend, clone 29F.1A12), CD44 (eBioscience, clone IM7), B220 (Biolegend, clone), CD19 (Biolegend, clone 1D3/CD19), FAS (BD Biosciences, clone Jo2), PNA (Vector Labs, clone FL-1071), Bcl-6 (BD Biosciences, clone K112-91) and FoxP3 (eBioscience, clone FJK-16s). Stainings of cell surface markers were performed in PBS containing 2% FBS. Staining for CXCR5 was described previously[16] . Briefly, cells were successively stained with purified anti-CXCR5 (BD Biosciences) for 1 hour at 4°C, biotinylated anti-rat IgG (Jackson Immunoresearch) for 30 min on ice and fluorescent-dye conjugated streptavidin (Thermo Fisher) for 30 min on ice. Stainings for FoxP3 and Bcl-6 were performed with the FoxP3/Transcription Factor Staining Buffer Set (eBioscience, 00-5523). Dead cells were stained using LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Life Technologies). Flow cytometry data were acquired in a FACSCanto II (BD Biosciences) or a FACSFortesa (BD Biosciences) and analyzed using FlowJo.
4
Comprehensive Immunophenotypic Analysis of Murine Splenocytes
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Splenocytes were stained with fluorophore-conjugated antibodies: CD19 (6D5 BioLegend catalog 115530), B220 (RA3-6B2 eBioscience catalog 48-0452-82), Bcl-6 (K112-91 BD Biosciences catalog 561525), CD4 (RM4-5 eBioscience catalog 56-0042-82), CD8a (53-6.7 BD Biosciences catalog 557959), CD25 (7D4 eBioscience catalog 13-0252-82), CD95 (Jo2 BD Biosciences catalog 554257), CD44 (IM7 eBioscience catalog 48-0441-80), CD62L (MEL-14 BD Biosciences catalog 553152), Foxp3 (FJK-16s eBioscience catalog 53-5773-82), IgM (II/41 eBioscience catalog 17-5790-82), and PD-1 (J43 eBioscience catalog 13-9985-81). Individual samples were acquired on a BD LSR Fortessa Flow Cytometer and analyzed with FlowJo software (TreeStar).
5
Quantifying T Cell Signaling Responses
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To examine the effect of IL-15 and IL-15TRANS at the protein level, primary T cells for each condition were harvested 2 days following IL-2, IL-15, or IL-15TRANS treatment. Antibodies used were as follows: pSTAT5 (611964, BD Biosciences), STAT5 (sc-482X, Santa Cruz), Bcl-6 (561520, BD Biosciences), pSTAT4 (5267S, Cell Signaling), STAT4 (sc-486X, Santa Cruz), Blimp-1 (A01647-40, Genscript), GAPDH (sc-25778, Santa Cruz), β-Actin (A00730-40, Genscript). Further details regarding the clone and/or dilution of antibodies used can be found in Supplementary Table S2 ).
6
Characterizing TH Subset Cells in Mice
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Joint draining LN cells from 6 week old KRN.g7, IDO1 ko KRN.g7, IDO2 ko KRN.g7, and dko KRN.g7 mice were harvested and stained for CD4+ T cells (BioLegend) and the following markers to distinguish TH subsets: bcl6 (BD Bioscience), foxP3 (Biolegend), gata3, rorγt, T-bet (all from eBioscience) as described (1 (link)). The samples were acquired on a BD FACSCanto II flow cytometer using FACSDiva Software and analyzed using FlowJo Software.
7
Immunoblot Analysis of Protein Expression
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An equal number of cells were collected and subjected to immunoblot analysis to determine protein expression levels of Bcl-6 (BD Biosciences, 561520, dilution 1:500), Blimp-1 (Genscript, A01647, dilution 1:500), STAT5 (Santa Cruz, sc-835, dilution 1:5,000), phospho-STAT5 (pY694, BD Biosciences, 611964, dilution 1:5,000) and V5-tagged proteins (Invitrogen, R960-25, dilution 1:5,000). In brief, separation of lysates by SDS–polyacrylamide gel electrophoresis was followed by immunoblot analysis. GAPDH (Santa Cruz, sc-25778, dilution 1:2,500) or β-actin (Genscript, A00730, dilution 1:10,000) expression was monitored to ensure equal protein loading. Additional information for antibodies can be found in Supplementary Table 3 and uncropped versions of all immunoblots are provided in Supplementary Fig. 5 .
8
Immunoblot Analysis of Cell Signaling
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Immunoblots were performed as described previously38 (link),77 (link). Briefly, cells were harvested and pellets were lysed and prepared via boiling for 15 min in 1X loading dye (50 mM Tris [pH 6.8], 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol). Lysates were separated by SDS-PAGE on 10% Bis-Tris Plus Bolt gels (ThermoFisher) and transferred onto 0.45 µm nitrocellulose membrane. Membranes were blocked with 2% nonfat dry milk in 1X TBST (10 mM Tris [pH 8], 150 mM NaCl, 0.05% Tween-20), and detection of indicated proteins was carried out using the following antibodies: Aiolos (39293, Active Motif, 1:20,000), Bcl-6 (clone K112, BD Biosciences, 1:500), pSTAT5(Y694/9) (clone 47 BD Biosciences, 1:5000), STAT5 (clone (D206Y, Cell Signaling, 1:5000), β-Actin (GenScript, 1:15,000), Eomes (Abcam, 1:5,000) goat anti-mouse:HRP (Jackson Immunoresearch, 1:5,000-1:30,000), mouse anti-rabbit:HRP (Santa Cruz, 1:5000-1:20,000).
9
Immunoblot Analysis of Immune Proteins
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Immunoblot analyses were performed as described previously44 (link). Antibodies used to detect chosen proteins were as follows: Bcl-6 (1:500, BD Pharmingen), T-bet (1:1,000, Santa Cruz), pSTAT3 Y705 (1:20,000, Abcam), STAT3 (1:5000, Santa Cruz), pSTAT4 Y693 (1:1000, Cell Signaling), and STAT4 (1:2500, BioLegend). For all experiments, β-actin (1:15,000, GenScript) expression was monitored to ensure equivalent protein loading. Original and uncropped images of immunoblots can be found in Supplementary Fig. 5 .
10
Multiparametric Flow Cytometry Panel
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Fluorescence-conjugated antibodies for CD4(Cat # 48-0042-82), CD8(Cat # 47-0081-82), CD44(Cat # 11-0441-85), CD45.1(Cat # 47-0453-82), CD45.2(Cat # 48-0454-82), Streptavidin(Cat # 25-4317-82), IgD(Cat # 48-5993-82), and Ki67(Cat # 25-5698-82) were purchased from eBioscience; CXCR5(Cat # 551961), CD138(Cat # 553713), IgG2α(Cat # 553894), PD1(Cat # 562671), Bcl-6(Cat # 561525), Fas(Cat # 554258), GL7(Cat # 553666), and H2A.X(pS139)(Cat # 564719) were purchased from BD Biosciences; B220(Cat # 103212), SLAM(Cat # 115904), IFNγ(Cat # 505810), and FITC Annexin V Apoptosis detection kit with 7-AAD(Cat # 640922) were purchased from Biolegend.
Anti-Ddb1 (cat #11380-1-AP), anti-β-Actin (cat #66009-1-Ig), and anti-ATR (cat #19787-1-AP) were purchased from Proteintech; anti-p53 (cat #2524), phosphor-Chk1 (S345) (cat #2348T), anti-cleaved-caspase8 (Asp387) (cat #8592S), anti-caspase8 (cat #4790T), anti-cleaved-caspase3 (Asp175) (cat #9664S), and anti-caspase3 (cat #9662) were purchased from Cell Signaling technology (CST); anti-Prkdc(cat #SC-390849) was purchased from SantaCruz; anti-ATM (cat #A19650) and anti-Chk1 (cat #A7653) were purchased from ABclonal Technology.
Anti-Ddb1 (cat #11380-1-AP), anti-β-Actin (cat #66009-1-Ig), and anti-ATR (cat #19787-1-AP) were purchased from Proteintech; anti-p53 (cat #2524), phosphor-Chk1 (S345) (cat #2348T), anti-cleaved-caspase8 (Asp387) (cat #8592S), anti-caspase8 (cat #4790T), anti-cleaved-caspase3 (Asp175) (cat #9664S), and anti-caspase3 (cat #9662) were purchased from Cell Signaling technology (CST); anti-Prkdc(cat #SC-390849) was purchased from SantaCruz; anti-ATM (cat #A19650) and anti-Chk1 (cat #A7653) were purchased from ABclonal Technology.
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