PBMCs were isolated from heparinized blood samples and stimulated with SARS-CoV-2 peptide pool or medium in the presence of 2 μM monensin (eBioscience, San Diego, Calif) for 24 hours. After culture, the cells were collected for flow cytometry analysis. Fluorescence-labeled mAbs against the following antigens were added to the cell suspensions as follows: fixable viability stain, CD3, CD4, CD45RA, CCR7, HLA-DR, PD-1, Tim-3, TIGIT, CTLA-4, and CD39 (BD Biosciences, San Jose, Calif). All these cell suspensions were incubated for 30 minutes on ice. In some experiments, cells were fixed and permeabilized, and stained with anti–IL-2, anti–IFN-γ, anti–TNF-α, anti–IL-4, and anti–IL-17 mAbs (eBioscience). Isotype controls with irrelevant specificities were included as negative controls. fixable viability stain was used to exclude dead cells from analysis. After washings, the pellets were resuspended in 300 μL staining buffer, followed by analysis with FACSCanto flow cytometer (BD Biosciences).
Tim 3
Manufactured by BD
Sourced in United States
TIM-3 is a laboratory equipment product manufactured by BD. It is a device used for the detection and analysis of specific proteins or molecules in biological samples. The core function of TIM-3 is to provide researchers and scientists with a tool for conducting various immunological and biochemical assays. The detailed specifications and intended use of this product are not available within the scope of this request.
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Lab products found in correlation
Lag 3, by BD (8 mentions)
Ctla 4, by BD (3 mentions)
Cd62l, by BD (3 mentions)
Ifn γ, by BD (3 mentions)
Pd l1, by BD (3 mentions)
Fixable viability stain, by BD (2 mentions)
Tigit, by BD (2 mentions)
Cd45ra, by BD (2 mentions)
Hla dr, by BD (2 mentions)
Nkg2d, by BD (2 mentions)
Perforin, by BD (2 mentions)
Anti il 2, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ, by Thermo Fisher Scientific (1 mentions)
Anti tnf α, by Thermo Fisher Scientific (1 mentions)
Anti il 4, by Thermo Fisher Scientific (1 mentions)
Anti il 17 mab, by Thermo Fisher Scientific (1 mentions)
Monensin, by Thermo Fisher Scientific (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Facs symphony, by BD (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
20 protocols using tim 3
1
SARS-CoV-2 Peptide Stimulation of PBMCs
2
Phenotypic Characterization of CAR T Cells
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All experiments were performed on a FACS Symphony (B.D. Biosciences), and data obtained were analyzed with FlowJo software (version 10.7.1). CAR expression was evaluated using a ROR1 biotinylated protein (ACRO Biosystem) and a streptavidin (PE-conjugated; B.D. biosciences). T cell profile and characterization were evaluated using mAb to CD45, CD3, CD4, CD8, CD62L, CCR7, PD-1, TIM-3, LAG-3, TIGIT, and the corresponding isotype controls (B.D. Biosciences). The presented results presenting CAR T cell phenotype data were performed using the following gating strategy: we excluded the doubles, by side scatter versus forward scatter, viability using LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen), followed by the populations CD45+, CD3+, CAR+ Cells (Supplementary Figure 1
3
Tumor Immune Cell Profiling Protocol
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Tumors were minced with a razor blade in RPMI media. Then, 50 µg/mL liberase (Sigma Aldrich Cat# 5401054001) was added for digestion and samples incubated for 20 min at 37 °C. The cell suspension was passed over a 100-micron filter and rinsed with 5 mL of RPMI. Samples were spun at 1500 rpm for 5 min. Red blood cells were lysed using ACK buffer. The PBMCs were treated with anti-CD16/CD32 (Fc block; BD Biosciences Cat# 553141) and the surface stained with fluorescently conjugated antibodies for FVS (BD Biosciences, #564406), CD3 (BD Biosciences Cat# 564010), CD4 (BD Biosciences Cat# 561830), CD8 (BD Biosciences Cat# 563046), PD-1 (BD Biosciences Cat# 748266), Tim-3 (BD Biosciences Cat# 747626), CD25 (BD Biosciences Cat# 563061), and FoxP3 (BD Biosciences Cat# 560403). LSRFortessa flow cytometer with FACSDiva software (BD Biosciences) was used for data acquisition and FlowJo Mac, version 10.8.1 software, was used for data analysis.
4
TGF-β1 and Galectin-9 Immune Modulation
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RPMI-1640 medium was purchased from Hyclone (Utah, USA) and Fetal Bovine Serum was from Bio International (Auckland, New Zealand). rhTGF-β1 and rhGalectin-9 were from R&D (Minneapolis, USA). Goat anti-human TGF-β1, goat anti-human Galectin-9 and Tim-3-Fc fusion protein were purchased from R&D Systems. Fluorescently-labeled antibody for FACS analysis, CD3, CD56, CD16, Tim-3, CD117, CD94, CD11b and CD69 were purchased from BD PharMingen (San Diego, California). CD107a was from Miltenyi Biotec.
5
Comprehensive NK Cell Phenotyping
6
Multi-panel Flow Cytometry Immunophenotyping
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Flow cytometry experiments were performed on Aurora (Cytek Biosciences), BD Symphony 3 Flow cytometer, BD FACS ARIA III Sorter, and BD Melody Cell Sorter from the flow core in the University of Pittsburgh or Center for Discovery and Innovation and analyzed by Flowjo (BD). CD45 (clone 30-F11), CD4 (clone RMT4-5), CD8a (clone 53.67), Tcf1/Tcf7 (clone C63D9), PD-1 (clone J43), Tim-3 (clone RMT3-23), Lag-3 (clone C9B7W), Thy1.2 (clone 53-2.1), IFN-γ (clone XMG1.2), Ly-6C (clone HK1.4), Ly-6G (clone 1A8), Sca-1 (clone D7), ST2 (clone RMST2-33), CD103 (clone M290), GzmB (clone QA16A02), Foxp3 (clone MF-14), Ki-67 (clone 16A8), CD11b (clone M1/70), CD11c (clone N418), CD86 (clone GL-1), MHCII (clone), F4/80 (clone BM8), CD206 (clone C068C2), Arg1 (clone A1exF5), CD25 (clone PC61), CD62L (clone MEL-14), CD44 (clone IM7), CD90.2 (clone 53-2.1), CD140a (clone APA5), LIVE/DEAD dye (Zombie NIR Dye) were purchased from BD Bioscience, Thermo Fisher Scientific, or BioLegend. For intracellular transcription factors and cytokines staining, cells were stimulated with a leukocyte activation cocktail (BD) for 6 hours and then followed the standard staining protocol described previously (49 ).
7
Multiparametric Flow Cytometry Analysis
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Antibodies specific to CD8 (MultiSciences, Hangzhou, Zhejiang, China), PD-1 (eBioscience, Ankara, TURKEY), and Tim-3 (BD Biosciences, San Jose, CA, USA) were used for surface staining of cells. All experiments were carried out with a CytoFLEX LX flow cytometer (BECKMAN COULTER, BREA, CA, USA), with a minimum of 1,000,000 events per sample. Calibrator beads were used to calibrate the FACS machine before each run. Cells were gated on live cells based on forward- and side-scatter properties. The samples were analyzed using FCS Express software version 7.0 (De Novo Software, Los Angeles, CA).
8
Multiparametric T Cell Phenotyping
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To evaluate frequencies of T cells expressing 1 or more checkpoint receptors and levels of T-cell activation (defined as percentage of T cells co-expressing HLA-DR and CD38) and cell cycling (Ki-67), cryopreserved PBMCs obtained at multiple timepoints underwent staining with antibodies for CD3, CD4, CD8, HLA-DR, CD38, Ki-67, PD-1, PD-L1, TIM3 (all BD Biosciences), CTLA4, LAG3, TIGIT (all Biolegend), and Live/Dead (Invitrogen). Cells were analyzed using BD LSRFortessa and FlowJo v10.8.
9
Multiparametric Flow Cytometry of T-Cell Subsets
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Surface staining was carried out for markers like CD3, CD8, and Tim-3 (using antibodies from BD Biosciences, Franklin Lakes, NJ, USA), followed by permeabilization and intracellular staining for FITC P24 (KC-57) (Beckman Coulter, Brea, CA, USA). The data analysis was completed by FlowJo version 8.0.3 after acquiring 50,000 gated events of lymphocytes on FACS Fusion I (BD Biosciences, USA).
10
Multiparameter Flow Cytometry Analysis
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We used the following fluorochrome-conjugated monoclonal antibodies: anti-human CD3, CD4, CD8, CD25, TIM-3, CD278, LAG-3, CTLA-4, CD134, CD137, CCR7, CD45RO, PD-1, PD-L1, CD20, CD56, CD14, CD33, CD11b, CD11c, CD16, CD80, CD206, and CD15 (BD Biosciences, Beckman Coulter, BioLegend). Cells were stained with these antibodies or the appropriate isotype control antibodies for 30 min at 4°C. We determined live/dead discrimination via exclusion of Fixable Viability Stain 780–positive cells (BD Biosciences). Stained cells were analyzed using BD Symphony (BD Biosciences) at the Flow Cytometry Core at Texas Children’s Hospital. We analyzed the data with Kaluza software (Beckman Coulter) and Cytobank (Cytobank Inc.) according to the manufacturer’s instructions.
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