Single-cell suspensions from the spleen and lymph nodes (LNs) were generated after mashing tissue through 70 μm cell strainer, and the cells were surface-, intracellularly or intranuclearly stained following standard protocols42 (link). The fluorochrome-conjugated antibodies, including anti-CD8 (53–6.7), anti-TCRβ (H57–597), anti-CD44 (IM7), and anti-CD62L (MEL-14), anti-CD45.2 (104), anti-KLRG1 (2F1), anti-IL-7Ra (A7R34), anti-Granzyme B (GB12), anti-IFN-g (XMG1.2), anti-TNF (MP6-XT22), anti-IL-2 (JES6–5H4), anti-hNGFR (ME20.4), were from eBiosciences, ThermoFisher Scientific. Anti-Tcf1 (C63D9) was from Cell Signaling Technology, and anti-Id3 (S30–778) was from BD Biosciences. For intranuclear detection of Id3 or Tcf1, surface-stained cells were fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBiosciences, ThermoFisher Scientific), followed by incubation with corresponding fluorochrome-conjugated antibodies. For cytokine staining, the cells were stimulated with GP33 (200 nM) in the presence of the protein transport inhibitor Brefeldin A for 5 hrs at 37°C, followed by standard intracellular staining. Cell sorting was performed on FACSAria (BD Biosciences). Data were collected on Fortessa LSR or FACSCelesta flow cytometers (BD Biosciences) and were analyzed with FlowJo software v10.7.1 (TreeStar).
Anti tcf1 c63d9
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-Tcf1 (C63D9) is a rabbit monoclonal antibody that recognizes the transcription factor Tcf1. It is designed for use in various immunodetection applications.
Automatically generated - may contain errors
Lab products found in correlation
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (6 mentions)
Facsaria, by BD (4 mentions)
Facsverse, by BD (3 mentions)
Fortessa lsr, by BD (2 mentions)
Anti id3 s30 778, by BD (2 mentions)
Facscelesta flow cytometer, by BD (2 mentions)
Anti il 2rβ tm β1, by BD (2 mentions)
Anti fasl mfl3, by BD (2 mentions)
Anti granzyme b gb 2, by Thermo Fisher Scientific (2 mentions)
Vβ tcr screening panel, by BD (2 mentions)
Anti foxp3, by Thermo Fisher Scientific (2 mentions)
Ionomycin, by BD (2 mentions)
Brdu staining kit, by Thermo Fisher Scientific (2 mentions)
Anti mouse perforin antibody s16009a, by BioLegend (2 mentions)
Anti human mouse granzyme b antibody gb11, by BioLegend (2 mentions)
A fixation permeabilization solutionkit, by BD (2 mentions)
Usingna ve cd8 t cell isolation kit, by STEMCELL (2 mentions)
Afacsaria, by BD (2 mentions)
Fortessa, by BD (2 mentions)
Annexin 5 apoptosis detection kit, by Thermo Fisher Scientific (2 mentions)
14 protocols using anti tcf1 c63d9
1
Single-Cell Immune Profiling in Lymphoid Tissues
2
Multiparametric Analysis of T Cell Populations
3
In Vivo Alpha4 Integrin Blocking Assay
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For in vivo α4 integrin blocking, anti-α4 integrin (PS/2) antibody was from Bio X Cell. Fluorescent dye–labeled antibodies specific for PD-1 (J43), CXCR5 (SPRCL5), CD8β (H35-17.2), CD45.1 (A20), CD45.2 (104), CD8α (53-6.7), CD44 (IM7), CX3CR1 (SA011F11), Granzyme A (GzA-3G8.5), integrin α4β7 (DAPK32), CXCR3 (CXCR3-173), CD39(24DMS1), CD73(TY/11.8), CD62L(MEL-14), Ly6C(HK1.4), CD49d (R1-2), integrin β1 (HMB1-1), integrin β7 (FIB504), IFN-γ (XMG1.2), IL-2 (JES6-5H4), Slamf6 (330-AJ), Tim-3 (RMT3-23), and Tox (TXRX10) were purchased from eBioscience, Biolegend, and Tonbo. Anti-CD16/32 (2.4G2) was produced in the lab and used in all FACS staining as FcR blocker. Anti–Tcf-1 (C63D9) and pSmad2/3 (EBF3R) were from Cell Signaling. Intranuclear staining for Tcf-1 and Tox was performed using a Foxp3 staining buffer set (Tonbo Bioscience). For intracellular cytokine staining, freshly isolated splenocytes were cultured with 0.1 μM GP33-41 peptide (AnaSpec) in the presence of Golgi Stop (BD) for 4–5 h at 37°C. After surface staining, IFN-γ and IL-2 were performed using permeabilization buffer (Invitrogen) following fixation. Ghost Dye Violet 510 (Tonbo Bioscience) was used to identify live cells. Washed and fixed samples were analyzed by BD LSRII or BD FACSCelesta and analyzed by FlowJo (TreeStar) software.
4
Phenotypic Analysis of Engineered T Cells
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Mononuclear cells were stained with fluorophore-conjugated monoclonal antibodies specific to murine CD45 (Ly5), Thy1.1 (OX-7), CD8α (53–6.7), PD-1 (J43), Granzyme B (NGZB), IFNγ (XMG1.2), TNFα (MP6-XT22), CD101 (Moushi101, eBiosciences), Slamf6 (330-AJ), Lag3 (C9B7W, Biolegend), KLRG1 (2F1, Biolegend) at a 1:100 dilution in PBS+2.5% FBS. Antibodies were purchased from BD Biosciences unless otherwise noted. To measure intracellular cytokine production, engineered T cells ± Msln406-414 peptide were incubated in vitro for 5 hours in the presence of GolgiPlug (BD Biosciences), stained for surface antigens, fixed and permeabilized (BD Biosciences Fixation/Permeabilization kit), and then stained with appropriate antibodies. For transcription factor analysis, cells were fixed using Foxp3 staining kit (Tonbo) for 30 min at 4°C, and stained with anti-Tox (TXRX10, Invitrogen) and anti-Tcf1 (C63D9, Cell signaling) at a 1:50 dilution in PBS+2.5% FBS overnight, and washed with perm/wash buffer. To quantify the frequency of cells proliferating, cells were surface stained as above followed by eBioscience Foxp3 Fixation/Permeabilization solutions prior to nuclear staining with anti-Ki67 (SolA15). Data were acquired on an LSRII, FacsCanto, or Fortessa and analyzed using FlowJo V.10.3 (BD Biosciences). Cell numbers infiltrating tissues were normalized to tumor weight.
5
Multiparametric Analysis of T Cell Populations
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Single cell suspension from the mouse thymus and spleen was prepared as previously described18 (link), 21 (link). The cells were surface-stained with various combinations of the following antibodies: anti-CD4 (M1/69), anti-CD8α (53-6.7), anti-TCRβ (H57–597), anti-CD69 (H1.2F3), anti-CD44 (IM7), anti-CD24 (M1/69), anti-CD62L (MEL-14), anti-CD40L (MR1) and CD45.2 (104) (all from eBiosciences). Anti-IL-2Rβ (TM-β1) and anti-FasL (MFL3) were from BD Biosciences. Anti-granzyme B (GB/2) was from Thermo Fisher Scientific. The Vβ TCR Screening Panel (BD Biosciences) was used to survey the Vβ TCR repertoire. For intranuclear staining, the surface-stained cells were fixed and permeabilized with Foxp3/transcription factor staining buffer set (eBiosciences), and stained with anti-Foxp3 (eBiosciences, FJK-16s), anti-Rorγt (eBiosciences, B2D), or anti-Tcf1 (C63D9, Cell Signaling Technologies). The stained cells were analyzed on FACSVerse (BD Biosciences). All antibodies were used at 1:50 to 1:100 dilution, and validated for mouse and flow cytometry as indicated by the data sheets from the manufacturers. The data were processed using FlowJo software (Version X, TreeStar). For cell sorting, surface-stained thymocytes or splenocytes were sorted on FACSAria (BD Biosciences) from Tcf7−/−Lef1−/− or control mice for RNA-Seq analysis and gene expression validation.
6
Characterization of CD8+ T Cell Phenotypes
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Single-cell suspensions were prepared from the spleen, LNs, and surface or intracellularly stained as described (Shan et al., 2022a (link)). The fluorochrome-conjugated antibodies were as follows: anti-CD8 (53–6.7), anti-TCRβ (H57-597), anti-CD45.2 (104), anti-Granzyme B (GB12), anti-IFN-γ (XMG1.2), anti-Tbet (4B10), anti-CD62L (MEL-14), anti-KLRG1 (2F1), anti-CD25 (PC61.5), anti-CD69 (H1.2F3), and anti-CD44 (IM7) were from Thermo Fisher Scientific; anti-Tcf1 (C63D9) from Cell Signaling Technology; anti-IL-7Rα (A7R34) and anti-CD45.1 (A20) were from BioLegend. For detection of Tcf1 and Tbet proteins, surface-stained cells were fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBiosciences, Thermo Fisher Scientific), followed by incubation with corresponding fluorochrome-conjugated antibodies. Apoptotic cells were detected with FLICA 660 Caspase-3/7 detection kit (Bio-Rad). Data were collected on FACSCelesta or FACSVerse (BD Biosciences) and were analyzed with FlowJo software V10.2 (TreeStar). For validation of CTCF deletion efficiency, cell lysates from sorted GFP+ naive or early effector CD8+ T cells were immunoblotted with anti-CTCF antibody (D31H2; Cell Signaling Technology) following standard protocols.
7
Immunofluorescence Analysis of Tissue Sections
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Tissue sections were incubated overnight with anti‐CD31 (D8V9E), anti‐α‐SMA (D4K9N), anti‐LEF1 (C12A5) and anti‐TCF1 (C63D9) antibodies (Cell Signaling Technology). After washing with PBS, the sections were incubated with secondary antibodies for 1 h. The stained sections were then exposed to DAPI for 10 min to stain the nuclei. Finally, the sections were observed and imaged using a fluorescence microscope.
8
Single-Cell Immune Profiling in Lymphoid Tissues
9
Multiparameter Flow Cytometry Analysis
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Cells were harvested, stained, acquired on a FACSCantoII (Becton Dickinson) and analyzed with FlowJo (Treestar). Dead cells were excluded using the Fixable Viability Dye eFluor506 (eBioscience). Antibodies conjugated to FITC, PE, PerCP Cy5.5, PE Cy7, APC, APC-Cy7 or Pacific Blue were purchased from BD Biosciences, eBioscience, BioLengend and R&D Systems. PE- or APC- conjugated mouse CD1d tetramers loaded with glycolipid PBS-57 (CD1d-tet) were obtained from the tetramer facility of the US National Institutes of Health. In brief, cells were incubated with FC block and stained with antibodies, and then fixed with 2% paraformaldehyde. For PLZF, T-bet, and TCF-1 intracellular staining, cells were permeabilized and stained with anti-PLZF (D-9) (Santa Cruz Biotechnology, Inc.) plus FITC anti-mouse (BD Biosciences), anti-T-bet PerCP-Cy5.5 (eBio4B10), both purchased from eBioscience, and with anti-TCF-1 (C63D9) (Cell Signaling) followed by goat anti-rabbit-Alexa647 (Invitrogen), using the Foxp3 Staining Buffer kit (eBioscience).
10
Phenotypic Analysis of Activated CD8+ T Cells
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