To analyze lymphoid populations within the tumor from one patient (patient 4), the fresh tumor tissue was finely minced with scissors and filtered through a nylon membrane, and a single-cell suspension was purified over a Ficoll–Hypaque density gradient. Cells were stained with the following anti-human antibodies: anti-αβTCR (IP26, eBioscience, San Diego, CA, USA), anti-CD8 (OKT8, eBioscience), anti-CD44 (IM7, eBioscience), anti-PD-1 (eBioJ105, eBioscience), anti-CD27 (O323, eBioscience), anti-CD20 (2H7, eBioscience), anti-CD1c (L161, eBioscience), anti-γδTCR (B1, BioLegend, San Diego, CA, USA), anti-CD4 (OKT4, BioLegend), anti-CD28 (CD28.2, BioLegend), anti-FAS (DX2, BioLegend), anti-ICOS (C398.4A, BioLegend), anti-IgM (MHM-88, BioLegend), anti-CD21 (B-ly4, BD Biosciences, San Jose, CA, USA), anti-CD23 (M-L233, BD Biosciences), and anti-IgD (Southern Biotech, Birmingham, AL, USA). Flow cytometry was performed using a BD FACS Canto II flow cytometer (BD Biosciences) and analyzed via FlowJo software (Tree Star, Ashland, OR, USA).
Anti cd44 im7
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-CD44 (IM7) is a monoclonal antibody that binds to the CD44 antigen, a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration. This antibody can be used for the detection and analysis of CD44-expressing cells in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (3 mentions)
Facsverse, by BD (3 mentions)
Facscelesta, by BD (2 mentions)
Anti cd4 okt4, by BioLegend (1 mentions)
Anti pd 1 ebioj105, by Thermo Fisher Scientific (1 mentions)
Anti icos c398.4a, by BioLegend (1 mentions)
Anti igd, by BD (1 mentions)
Anti cd8 okt8, by Thermo Fisher Scientific (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Anti cd40 1c10, by Thermo Fisher Scientific (1 mentions)
Anti cd28 37.51, by BD (1 mentions)
Control rat igg, by Innovative Research (1 mentions)
Anti cd3 2c11, by BD (1 mentions)
Facsaria fusion cell sorter, by BD (1 mentions)
Anti cd62l mel 14, by BD (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Anti cd4 rm4 5, by Thermo Fisher Scientific (1 mentions)
Fjk 16, by Thermo Fisher Scientific (1 mentions)
Anti cd11c n418, by Thermo Fisher Scientific (1 mentions)
Anti cd4 gk1, by Cytek Biosciences (1 mentions)
7 protocols using anti cd44 im7
1
Profiling Tumor-Infiltrating Lymphocytes
2
Murine Immune Cell Phenotyping
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For flow cytometric analysis and sorting, the following antibodies were purchased from BD Biosciences: anti-CD4 (RM4-5), anti-CD8a (53-6.7), anti-CD11c (HL3), anti-CD25 (PC61), anti-I-A/I-E (MHC class II, 2G9), anti-DO11.10 TCR (KJ1.26), anti-CD16/CD32 (2.4G2, for blocking Fc receptors) and anti-Thy1.1 (CD90.1, OX-7). The following antibodies were purchased from eBioscience: anti-CD80 (16-10A1), anti-CD86 (GL1), anti-LFA-1 (CD11a, M17/4), anti-ICAM-1 (CD54, YN1/1.7.4), anti-CD44 (IM7) and anti-Thy1.2 (CD90.2, 53-2.1). Anti-CD45RB (C363-16A) was purchased from BioLegend (San Diego, CA, USA). For reagents used in culture, anti-CD28 (37.51) and anti-CD3 (2C11) antibodies were purchased from BD Biosciences, and anti-CD40 (1C10) from eBioscience. For in vivo injections, anti-Thy1.2 (CD90.2, 30-H12) was purchased from BD Biosciences and also purified from ascites with hybridoma cells. Control rat IgG was purchased from Molecular Innovations (Novi, MI, USA).
3
Isolation of Antigen-Specific T Cells
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At day 50 or 100 after primary vaccination, or at day 10 after secondary vaccination, mice were sacrificed, spleens were isolated and passed through 70 μm cell strainer (BD Falcon). After erythrocyte lysis, cell suspensions were washed, stained with anti-CD8α mAb (53-6.7, eBioscience), anti-CD44 (IM7, eBioscience) and anti-CD62L (MEL-14, BD Pharmingen) and PE-conjugated H-2Db/E749-57 or H-2Kb/OVA257-264 tetramers and sorted on a BD FACSAria Fusion cell sorter. Live cells were selected based on propidium iodide exclusion. All samples were maintained at 4oC for the duration of the sort, and purity was at 95%-98% for all samples. Isolated cells were subsequently used for in vivo, in vitro, RNAseq or ChIPseq assays.
4
Comprehensive Antibody Panel for Murine T-cell Analysis
5
Flow Cytometric Analysis of Spleen Cells
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
Multi-parameter Flow Cytometry Analysis
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