For intracellular cytokine staining, cells were stimulated with Cell Stimulation Cocktail plus protein transport inhibitors (eBioscience, San Diego, CA) for 4 – 6 hours. Surface antigens were stained with the following antibodies: anti-CD4 (BD Biosciences, RM4-5), anti-CD44 (BD Biosciences, IM7), anti-CD62L (eBioscience, MEL-14), anti-CD45.1, and anti-CD25 (BD Biosciences, 7D4). For intracellular staining, cells were fixed and permeabilized with the Foxp3 staining buffer kit (eBioscience) and stained for intracellular cytokines and proteins with anti-IFN-γ (BD Biosciences, XMG1.2), anti-IL-17 (BioLegend, TC11-18H10.1), anti-CTLA-4, and anti-Foxp3 (eBioscience, FJK-16s) antibodies. Flow cytometric analyses were performed on a FACSCalibur instrument (BD Biosciences) and analyzed using FlowJo software (TreeStar, Ashland, OR).
Anti ctla 4
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-CTLA-4 product is a laboratory reagent used in research applications. It functions as an antibody that binds to the CTLA-4 protein, which plays a role in regulating the immune system. This product can be utilized in various immunological studies and assays, but its specific intended use should be determined by the researcher.
Automatically generated - may contain errors
Lab products found in correlation
Anti foxp3, by Thermo Fisher Scientific (4 mentions)
Anti cd62l, by Thermo Fisher Scientific (2 mentions)
Anti cd4, by BD (2 mentions)
Anti cd45, by Thermo Fisher Scientific (2 mentions)
Anti ki67, by Thermo Fisher Scientific (2 mentions)
Anti pd 1, by Thermo Fisher Scientific (2 mentions)
Anti cd11b, by BioLegend (2 mentions)
Anti cd8, by BioLegend (2 mentions)
Cd103 apc, by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Foxp3 staining buffer kit, by Thermo Fisher Scientific (1 mentions)
Cell stimulation cocktail plus protein transport inhibitor, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ, by BD (1 mentions)
Anti cd44, by BD (1 mentions)
Anti il 17, by BioLegend (1 mentions)
Anti cd45, by BD (1 mentions)
Facscalibur instrument, by BD (1 mentions)
Anti cd45ra apc, by BioLegend (1 mentions)
Pe cy7 anti cd127, by Sony (1 mentions)
Anti pd 1 apc, by BioLegend (1 mentions)
7 protocols using anti ctla 4
1
Intracellular Cytokine Staining for Immune Cell Analysis
2
Multiparametric Flow Cytometry Panel for T Cell Phenotyping
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For the sorting experiments, FITC-coupled anti-CD4 and PE-coupled anti-CD25 were purchased from BD Biosciences (MountainView, CA, USA), APC anti-CD45RA was purchased from BioLegend (San Diego, CA, USA), PE-Cy5 anti-CD11c was purchased from Beckman Coulter (Fullerton, CA, USA), BV650 anti-CD3, PE-Cy7 anti-CD127 and BV510 anti-CD16 were purchased from Sony Biotechnology (San Jose, CA, USA), and Violet Blue anti-CD14 was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). For the proliferation assays, SEE was purchased from Toxin Technology Inc (Sarasota, FL, USA), CellTrace™ carboxyfluorescein succinimidyl ester (CFSE) was purchased from Molecular Probes (Eugene, OR, USA) and CellTrace™ Violet (CTV) was purchased from Invitrogen (Carlsbad, CA, USA). For phenotyping, PE-anti-PD-L1, PE-anti-BTLA, PE-anti-LAG3, PE-anti-TIM3, APC-anti-PD-1, and APC-anti-4-1BB were purchased from BioLegend (San Diego, CA, USA), APC anti-ICOS was purchased from Invitrogen (Carlsbad, CA, USA) and APC anti-TIGIT was purchased from Sony Biotechnology (San Jose, CA, USA). PE-anti-PD-L2 and anti-CTLA-4 were purchased from ebiosciences (San Diego, CA, USA).
3
Lung and Tumor Immune Cell Isolation
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Lymphocytes were isolated from lung and tumor tissue by digestion with collagenase A (1 mg/ml; Roche) and DNase I (0.5 µg/ml; Roche) in isolation buffer (RPMI 1640 supplemented with 5% FBS, 1% l -glutamine, 1% penicillin-streptomycin, and 10 mM Hepes) for 30 min at 37°C. Cells were filtered through 100-µm cell strainers, washed in isolation buffer, and stained in PBS supplemented with 0.25% BSA, 2 mM EDTA, and 0.1% sodium azide. Antibodies used included anti-CD45, anti-Foxp3, anti–IL-18Rα, anti-ST2, anti-CD62L, anti-CD103, anti–PD-1, anti-GITR, anti–CTLA-4, anti-KLRG1, anti-Ki67, anti-CD5, anti-NK1.1, anti-CD45R (B220), anti–IL-17, anti–IL-4, anti-IFNγ, and anti–Ly-6C (eBioscience); anti-TCRβ, anti-CD3, anti-CD4, anti-CD8, anti-CD127, anti-CD11B, anti-MHCII, and anti-Gr1 (BioLegend); anti-CD44 and anti-CD25 (Tonbo); anti–IL-5 and anti-TNFα (BD PharMingen); and anti-AREG (R&D Systems). For exclusion of dead cells, samples were first stained with Ghost Dye (Tonbo) cell viability reagent. Intracellular staining for cytokines, Foxp3, AREG, Ki-67, and CTLA-4 was performed using the Foxp3/transcription factor staining buffer set (eBioscience) as per manufacturer’s protocol. The H2-Kb OVA257–264 tetramer was obtained from the NIH Tetramer Core Facility.
4
Comprehensive Immune Profiling of Tumor-Bearing Mice
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The following Abs were used: anti-CTLA-4 (HMCD15201, Thermo Fisher), anti-CD39 (143804, BioLegend), anti-CD8 (553031, BioLegend), anti-CD73 (127220, BioLegend), anti-Tbet (644810, BioLegend), anti-CD44 (103030, BioLegend), anti-KLRG1 (138414, BioLegend), anti-CD11b (101230, BioLegend), anti-CXCR5 (551961, BD Biosciences), anti-CD25 (564571, BD Biosciences), anti-CD4 (553052, BD Biosciences), anti-CD107a/b (553793/558758, BD Biosciences), anti-B220 (561102, BD Biosciences), anti-PD1 (11-9985-81, eBioscience), anti-Foxp3 (50-5773-82, eBioscience), anti-CD11c (17-0114-82, eBioscience), anti-CD45 (12-0451-82, eBioscience), anti-CD49b (25-5971-82, eBioscience) and anti-TCF-1 (2206S, Cell Signaling Technology). Fixable Viability Dye (65-0865-14, eBioscience)-stained cells were excluded from analysis. Tumor-bearing mice were sacrificed; the tumors were sliced and digested with type I collagenase (A004194-0001, Sangon Biotech) for 1 hour at 37°C; and the spleens and dLNs were ground to generate single-cell suspensions. The single-cell suspensions were filtered through 70 µm strainers (352350, BD Biosciences) and stained as described. The stained cells were evaluated by BD FACS Canto II flow cytometry, and the flow cytometry data were analyzed with FlowJo software (Tree Star).
5
OX40L-OX40 Modulation of T Cell Immunity
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Recombinant mouse GM-CSF was purchased from Miltenyi Biotec (Auburn, CA). Recombinant mouse FLT3L was purchased from Gemini Bio-Products (West Sacramento, CA). CellTrace Violet cell proliferation kit was purchased from ThermoFisher Scientific (Waltham, MA). Anti-FOXP3, anti-CD4, anti-OX40L, anti-CD11c, anti-Helios, anti-CTLA4, anti-CD39, anti-CD11b, anti-Sirpα, anti-CD80, anti-CD86, anti-CD274, anti-CD273, anti-MHCII, anti-Ki67, and anti-MGL2 fluorescently coupled antibodies were purchased from ThermoFisher Scientific (Waltham, MA). Foxp3/Transcription Factor Staining Buffer Kit was purchased from Tonbo Biosciences (San Diego, CA). Purified anti-OX40L (RML134L) was purchased from Biolegend (San Diego, CA) and purified anti-OX40 agonist (OX-86) was purchased from ThermoFisher Scientific (Waltham, MA).
6
Comprehensive Murine and Human Immune Profiling
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Flow cytometry antibodies for murine samples included CD3e-PE, PD1-BV605, CD45-BV786 (BDbiosciences), CD90.1-FITC, CD62L-PECy7, CD45.1-PE (ebioscience), CD45.2-Alexa700, CD-8α-Percp Cy5.5, CD4-FITC, CD103-APC, CD24-APCcy7 (Invitrogen), CD44-APCy7, CD4-BV421, F4/80-PercpCy5.5, CD39-PECy7, PDL1-PE, CD90.2-Alexa 700, MHCII-BV421, CD11b-BV650, Ly6C-BV711 (Biolegend), CD8α-PE-Texas red (life technologies), CD278 (ICOS)-PE (Biolegend). Flow cytometry antibodies for human samples included CD45-BV510, CD3-Alexa700, CD4-BV785, CD8-BV711, CTLA-4-PE/Dazzle 594, 4-1BB-PE (Biolegend), CD39-BV650, PD-1-PE-Cy7 and Ki-67 Alexa 488 (BD Biosciences), CD103-APC and FOXP3-Alexa 700 (eBioscience).
Mouse antibodies for the explant assays included anti-PD1 (RMP1-14) and anti-CTLA-4 (9D9) by BioXCell and OX40 (OX86) kindly provided by Dr. Andrew Weinberg (EACRI). Human antibodies for the explant assays included anti-PD1 and anti-CTLA4 purchased from Invitrogen and anti-OX40 kindly provided by Dr. Andrew Weinberg (EACRI).
Mouse antibodies for the explant assays included anti-PD1 (RMP1-14) and anti-CTLA-4 (9D9) by BioXCell and OX40 (OX86) kindly provided by Dr. Andrew Weinberg (EACRI). Human antibodies for the explant assays included anti-PD1 and anti-CTLA4 purchased from Invitrogen and anti-OX40 kindly provided by Dr. Andrew Weinberg (EACRI).
7
Immunofluorescence Analysis of Immune Markers in Gastric Cancer Mucosa
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Mucosa from GC patients was fixed in 10% formalin and embedded in paraffin. Paraffin-embedded sections were probed with anti-CD4+ (Novus Biologicals, Littleton, CO, USA), anti-CD8+ (Novus Biologicals), anti-PD-L1 (Invitrogen, Carlsbad, CA, USA), and anti-CTLA-4 (Invitrogen) primary antibodies at 4°C overnight. They were then stained with secondary antibodies conjugated with fluorescein isothiocyanate (Santa Cruz Biotechnology, Santa Cruz, CA, USA), allophycocyanin (Invitrogen), and phycoerythrin (Southern Biotech, Birmingham, AL, USA) at room temperature for 2 h. Nuclei were stained with 4,’6-diamidino-2-phenylindole (DAPI; Invitrogen). Immunofluorescence images were obtained using an LSM 700 confocal microscope (Zeiss, Oberkochen, Germany) at 200× magnification. Images were analyzed using ZEN 2 (blue edition) (Zeiss).
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