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Lag 3

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LAG-3 is a laboratory instrument designed for the measurement and analysis of various parameters. It is a core piece of equipment used in research and diagnostic settings. The LAG-3 provides reliable and consistent data to support scientific investigations and clinical applications.

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Lab products found in correlation

Tim 3, by BD (8 mentions) Cd62l, by BD (6 mentions) Tim 3, by BioLegend (4 mentions) Ifn γ, by BD (4 mentions) Granzyme b, by BD (3 mentions) Tnf α, by BD (3 mentions) Pd l1, by BD (2 mentions) Biotinylated cetuximab, by Bristol-Myers Squibb (2 mentions) Cd171, by Miltenyi Biotec (2 mentions) Pdcd1, by BioLegend (2 mentions) Fortessa x20, by BD (2 mentions) Annexin 5, by Santa Cruz Biotechnology (2 mentions) Bcl 2 bcl 10c4, by BioLegend (2 mentions) Cd101, by BioLegend (2 mentions) 7 amino actinomycin, by BD (2 mentions) Bcl xl h 5, by Santa Cruz Biotechnology (2 mentions) Ctla 4, by Cytek Biosciences (2 mentions) Lag 3, by Enzo Life Sciences (2 mentions) Cytofix cytoperm, by BD (2 mentions) Cd62l, by BioLegend (2 mentions)

Close spelling variants of this product

Anti-mouse Lag-3 (1 citations)

22 protocols using lag 3

1

Investigating SRSF2 Function in Tumor-Infiltrating Lymphocyte Immune Response

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To investigate the functions of SRSF2 in the immune response of TILs against autologous tumor cell lines, TILs transfected with SRSF2 siRNA were co-cultured with autologous tumor cell lines. Then, flow cytometry was performed with an anti-CD8 antibody (Biolegend, 344704) and anti-IFN-γ antibody (BD Biosciences, 554552) on BD AccuriC6 (BD Biosciences). To examine the expression level of the immune checkpoints in the TILs and PBMCs, the frequency of immune checkpoint + and CD8 + TILs was assessed using flow cytometry on BD AccuriC6 (BD Biosciences). The antibodies against the immune checkpoints used for flow cytometry included PD-L1 (BD Biosciences, 561787), BTLA (BD Biosciences, 564802), CTLA-4 (BD Biosciences, 561717), LAG3 (BD Biosciences, 565617), and CD160 (BD Biosciences, 562351). All data were analyzed by FlowJo software.
Wang Z., Li K., Chen W., Wang X., Huang Y., Wang W., Wu W., Cai Z, & Huang W. (2019). Modulation of SRSF2 expression reverses the exhaustion of TILs via the epigenetic regulation of immune checkpoint molecules. Cellular and Molecular Life Sciences, 77(17), 3441-3452.
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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).
Moreno-Cortes E., Franco-Fuquen P., Garcia-Robledo J.E., Forero J., Booth N, & Castro J.E. (2023). ICOS and OX40 tandem co-stimulation enhances CAR T-cell cytotoxicity and promotes T-cell persistence phenotype. Frontiers in Oncology, 13, 1200914.
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3

Comprehensive Immune Profiling of T Cells

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Cell-surface expression of CD4 (BD Biosciences), CD8 (BioLegend), and CD171 (cat#130-100-691, Miltenyi Biotec) was detected by fluorophore-conjugated monoclonal antibodies. EGFRt expression was detected using biotinylated cetuximab (Bristol-Myers Squibb) and a phycoerythrin (PE)-conjugated streptavidin antibody (BioLegend). T cell activation and exhaustion were assessed by fluorophore-conjugated monoclonal antibodies detecting CD137 (BioLegend), CD25 (BioLegend), PD-1 (also known as PDCD1 or CD279, BioLegend), TIM3 (Biolegend), and LAG3 (BD Biosciences). Flow cytometry was performed on a Fortessa X-20 (BD Biosciences) and data were processed using FlowJo software (Tree Star Inc.). Dead cells were excluded from analyses using LIVE/DEADTM Fixable Green Dead Cell Stain Kit (Life Technologies).
Ali S., Toews K., Schwiebert S., Klaus A., Winkler A., Grunewald L., Oevermann L., Deubzer H.E., Tüns A., Jensen M.C., Henssen A.G., Eggert A., Schulte J.H., Schwich E., Rebmann V., Schramm A, & Künkele A. (2020). Tumor-Derived Extracellular Vesicles Impair CD171-Specific CD4+ CAR T Cell Efficacy. Frontiers in Immunology, 11, 531.
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4

Comprehensive Antibody Panel for Mouse and Human T Cell Phenotyping

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For mouse studies, the following antibodies were purchased: from BioLegend: 2B4 (m2B4), BCL-2 (BCL/10C4), CD101 (Moushi101), CD11c (N418), CD127 (A7R34), CD19 (6D5), CD25 (PC61.5), CD3 (145–2C11), CD38 (90), CD39 (Duha59), CD40 (3/23), CD44 (IM7), CD62L (MEL-14), CD69 (H1.2F3), CD70 (FR70), CD80 (16–10A1), CD86 (GL-1), CD90.1 (OX-7 and HIS51), CD90.2 (30-H12 and 53–2.1), CXCR5 (L138D7), Eomes (Dan11mag), GZMB (GB11), IFNγ (XMG1.2), IL-2 (JES6–5H4), KLRG1 (2F1), LAG-3 (C9B7W), MHC I-A/I-E (M5/114.15.2), PD-1 (RMP1–30), T-bet (4B10), TIM-3 (RMT3–23), TNF (MP6-XT22), and 7-amino-actinomycin (7-AAD); from BD Biosciences: annexin V, CD95 (Jo2), Ki67 (B56), Vb7 (TR310); BCL-xL (H-5; Santa Cruz Biotechnology); BIM (C34C5; Cell Signaling Technology), CD8 (53–6.7; eBioscience), CTLA-4 (UC10–410-11; Tonbo Biosciences), TCF-1 (C63D9; Cell Signaling Technology), TIGIT (GIGD7; eBioscience).
For human studies, the following antibodies were purchased: CD39 (A1; BioLegend), CD45RA (HI100; BioLegend), CD45RO (UCHL1; BioLegend), CD8 (RPA-T8; BioLegend), LAG-3 (17B4; Enzo Life Sciences), PD-1 (EH12.1; BD Biosciences) and TIM-3 (F38–2E2; BioLegend).
For flow cytometric detection and analysis of mouse and human TOX, anti-human/mouse TOX antibody clone REA473 was used (Miltenyi Biotec); antibody clone REA293 was used as TOX isotype (Miltenyi Biotec).
Scott A.C., Dündar F., Zumbo P., Chandran S.S., Klebanoff C.A., Shakiba M., Trivedi P., Menocal L., Appleby H., Camara S., Zamarin D., Walther T., Snyder A., Femia M.R., Comen E.A., Wen H.Y., Hellmann M.D., Anandasabapathy N., Liu Y., Altorki N.K., Lauer P., Levy O., Glickman M.S., Kaye J., Betel D., Philip M, & Schietinger A. (2019). TOX is a critical regulator of tumour-specific T cell differentiation. Nature, 571(7764), 270-274.
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5

Multiplex Analysis of Tumor-Immune Interactions

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The details of co-culture assays are included in the Supplementary Methods Appendix. Human TNF-α, IFN-g and IL-2 were measured using a Luminex xMAP Multiplex Assay with Millipore Sigma antibodies (Burlington, MA, USA). T cell “exhaustion” markers were detected using human TIM-3 (Biolegend, San Diego CA, USA), LAG 3 (BD Biosciences, San Jose, CA, USA) and PD-1 (Biolegend, San Diego CA, USA) antibodies. We measured pCD3z activity in a 5-hour short-term cytotoxicity assay by expression of phospho-CD247 by flow cytometry and isotype control background was subtracted (Invitrogen, Waltham, MA, USA). The details of the western blot assay are included in the Supplementary Methods Appendix.
Atilla P.A., McKenna M.K., Watanabe N., Mamonkin M., Brenner M.K, & Atilla E. (2021). Combinatorial Antigen Targeting Strategies for Acute Leukemia: Application to Myeloid Malignancy. Cytotherapy, 24(3), 282-290.
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6

Multiparametric Flow Cytometry Analysis of Retinoblastoma Cells

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Cell-surface expression of GD2 (cat#565991, BD Biosciences), CD8 (cat#301041, BioLegend) and CD171 (cat#130–100-691, Miltenyi Biotec) was detected by fluorophore-conjugated monoclonal antibodies. EGFRt expression was detected using biotinylated cetuximab (Bristol-Myers Squibb) and a phycoerythrin (PE)-conjugated streptavidin antibody (cat#12–4317-87, BioLegend). Activation and exhaustion were assessed by fluorophore-conjugated monoclonal antibodies detecting CD137 (cat#309819, BioLegend), CD25 (cat#302622, BioLegend), PD1 (also known as PDCD1 or CD279, cat#329922, BioLegend), TIM3 (cat#345006, Biolegend) and LAG3 (cat#565721, BD Biosciences). Flow cytometry was performed on a Fortessa X-20 (BD Biosciences) and data processed using FlowJo software (Tree Star Inc.). Dead cells were excluded from analyses using LIVE/DEAD™ Fixable Green Dead Cell Stain Kit (cat#L23101, Life Technologies). QuantiBRITE PE calibration beads (BD Biosciences) were used to determine GD2 and CD171 antigen density on retinoblastoma cells according to the manufacturer’s instructions.
Andersch L., Radke J., Klaus A., Schwiebert S., Winkler A., Schumann E., Grunewald L., Zirngibl F., Flemmig C., Jensen M.C., Rossig C., Joussen A., Henssen A., Eggert A., Schulte J.H, & Künkele A. (2019). CD171- and GD2-specific CAR-T cells potently target retinoblastoma cells in preclinical in vitro testing. BMC Cancer, 19, 895.
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7

Isolation and Characterization of Tumor-Infiltrating Immune Cells

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Tumors were cut into small pieces and incubated in DMEM medium +10% FCS + SC + 0.8mg/mL Collagenase type I (Worthington) + 60U/mL of DNase on a 37°C shaker at 300RPM. After 45–60 min, the tissue was mashed through a 70μm cell strainer using the plunger flange of a syringe and cell suspensions treated with ACK lysis solution to remove any erythrocytes. Cells were labeled with the following fluorescently-labeled mAb: CD8α (53–6.7), CD69 (H1.2F3), PD-1 (J43), LAG-3 (eBioC9B7W), IFN-γ (XMG1.2), CD45.2 (104), Thy1.1 (HIS51), Ki67 (51-36524X BD). Samples requiring intracellular labeling were subsequently treated with Cytofix/Cytoperm for 10 min at 4°C and washed in Perm/Wash (BD Biosciences) before labeling. For Ki67 analysis, Foxp3 staining fixation and permeabilization set (eBioscience) was used. CFSE labeling was performed as previously described (13 (link)). Samples were run on a FACSCanto flow cytometer (BD Biosciences) and analyzed using FlowJo software.
Danahy D.B., Jensen I.J., Griffith T.S, & Badovinac V.P. (2019). Polymicrobial sepsis has the capacity to reinvigorate tumor-infiltrating CD8 T cells and prolong host survival. Journal of immunology (Baltimore, Md. : 1950), 202(10), 2843-2848.
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8

Multiparametric Flow Cytometry Profiling

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mAbs for human CD3 (APC-H7; SK7; 560176), CD4 (BV711; SK3; 563028), CD8 (APC; SK1; 340584), CD45RA (PE; HI100; 555489), CD45RO (BV786; UCHL1; 564290), CD69 (FITC; L78; 347823), CCR7 (FITC; 150503; FAB197F-100), PD-1(PE-Cy7; EH12.1;561272), Lag3(PE;T47-530;565616), FLAG (APC; L5; 637308), Granzyme-B (PE;GB11;561142) from BD biosciences and BioLegend were used. Samples were acquired with BD FACSCanto II or BD LSRFortessa. A minimum of 10 000 events were acquired for each sample and were analyzed using FlowJo 10 (FlowJo).
Wang G., Zhou X., Fucà G., Dukhovlinova E., Shou P., Li H., Johnston C., Mcguinness B., Dotti G, & Du H. (2021). Fully human antibody VH domains to generate mono and bispecific CAR to target solid tumors. Journal for Immunotherapy of Cancer, 9(4), e002173.
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9

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.
Wang D., Porter C.E., Lim B., Rosewell Shaw A., Robertson C.S., Woods M.L., Xu Y., Biegert G.G., Morita D., Wang T., Grilley B.J., Heslop H., Brenner M.K, & Suzuki M. (2023). Ultralow-dose binary oncolytic/helper-dependent adenovirus promotes antitumor activity in preclinical and clinical studies. Science Advances, 9(13), eade6790.
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10

Comprehensive Antibody Panel for Mouse and Human T Cell Phenotyping

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For mouse studies, the following antibodies were purchased: from BioLegend: 2B4 (m2B4), BCL-2 (BCL/10C4), CD101 (Moushi101), CD11c (N418), CD127 (A7R34), CD19 (6D5), CD25 (PC61.5), CD3 (145–2C11), CD38 (90), CD39 (Duha59), CD40 (3/23), CD44 (IM7), CD62L (MEL-14), CD69 (H1.2F3), CD70 (FR70), CD80 (16–10A1), CD86 (GL-1), CD90.1 (OX-7 and HIS51), CD90.2 (30-H12 and 53–2.1), CXCR5 (L138D7), Eomes (Dan11mag), GZMB (GB11), IFNγ (XMG1.2), IL-2 (JES6–5H4), KLRG1 (2F1), LAG-3 (C9B7W), MHC I-A/I-E (M5/114.15.2), PD-1 (RMP1–30), T-bet (4B10), TIM-3 (RMT3–23), TNF (MP6-XT22), and 7-amino-actinomycin (7-AAD); from BD Biosciences: annexin V, CD95 (Jo2), Ki67 (B56), Vb7 (TR310); BCL-xL (H-5; Santa Cruz Biotechnology); BIM (C34C5; Cell Signaling Technology), CD8 (53–6.7; eBioscience), CTLA-4 (UC10–410-11; Tonbo Biosciences), TCF-1 (C63D9; Cell Signaling Technology), TIGIT (GIGD7; eBioscience).
For human studies, the following antibodies were purchased: CD39 (A1; BioLegend), CD45RA (HI100; BioLegend), CD45RO (UCHL1; BioLegend), CD8 (RPA-T8; BioLegend), LAG-3 (17B4; Enzo Life Sciences), PD-1 (EH12.1; BD Biosciences) and TIM-3 (F38–2E2; BioLegend).
For flow cytometric detection and analysis of mouse and human TOX, anti-human/mouse TOX antibody clone REA473 was used (Miltenyi Biotec); antibody clone REA293 was used as TOX isotype (Miltenyi Biotec).
Scott A.C., Dündar F., Zumbo P., Chandran S.S., Klebanoff C.A., Shakiba M., Trivedi P., Menocal L., Appleby H., Camara S., Zamarin D., Walther T., Snyder A., Femia M.R., Comen E.A., Wen H.Y., Hellmann M.D., Anandasabapathy N., Liu Y., Altorki N.K., Lauer P., Levy O., Glickman M.S., Kaye J., Betel D., Philip M, & Schietinger A. (2019). TOX is a critical regulator of tumour-specific T cell differentiation. Nature, 571(7764), 270-274.
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