Lag3 bv711

Manufactured by BD

LAG3-BV711 is a fluorochrome-conjugated antibody that binds to the Lymphocyte Activation Gene 3 (LAG3) protein. LAG3 is an inhibitory receptor expressed on the surface of activated T cells, B cells, and natural killer cells. The BV711 fluorochrome is used for flow cytometry and other fluorescence-based applications.

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Lab products found in correlation

Pd1 pecf594, by BD (4 mentions) Cd4 buv395, by BD (4 mentions) 41bb percpef710, by Thermo Fisher Scientific (4 mentions) Live dead ghost dye 780, by Cytek Biosciences (4 mentions) Cd3 fitc, by BD (4 mentions) Cd8 buv805, by BD (4 mentions) Tim3 apc, by Thermo Fisher Scientific (3 mentions) Ctla 4 pe, by Thermo Fisher Scientific (2 mentions) Immunomagnetic negative selection, by STEMCELL (1 mentions) Rainbow beads, by Spherotech (1 mentions) Tigit bv421, by BD (1 mentions) Cd25 bv786, by BD (1 mentions) Cd8 apc h7, by BD (1 mentions) Cd4 bv605, by BD (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions) Pd 1 apc, by BD (1 mentions) Brilliant stain buffer, by BD (1 mentions) Cytofix, by BD (1 mentions) Il 2 apc, by Thermo Fisher Scientific (1 mentions) Tnf α pe cy7, by BD (1 mentions)

5 protocols using lag3 bv711

Adoptive Transfer of OT-1 T Cells

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For adoptive transfer of OT-1 T cells into B6 mice, OT-1 splenocytes were harvested as described above. CD8 T cells were isolated using immunomagnetic negative selection (StemCell, Vancouver, Canada; 19853), rinsed and suspended in PBS, and 2 × 10⁶ cells were adoptively transferred into 6–10 wk old, female, B6 mice via intraperitoneal injection. The day following transfer, mice were immunized subcutaneously with an individual SIINFEKL APL (100 µg) in complete Freund’s adjuvant (Sigma, F5881) or vehicle. Mice were euthanized at the times indicated, spleens were collected, processed as described above, and analyzed via flow cytometry using the following antibodies: SIINFEKL H2K^b tetramer-BV421 (NIH Tetramer Core Facility), CD3-FITC (BD 555274), CD4-BUV395 (BD 563790), CD8-BUV805 (BD 564920), LAG3-BV711 (BD 563179), PD1-PECF594 (BD 562523), TIM3-APC (eBioscience 17-5871-82), 41BB-PerCPeF710 (eBioscience 46-1371-82), CTLA4-PE (eBioscience 12-1529-42), CD44-BV786 (BD 563736) and Live/Dead Ghost dye 780 (Tonbo 13-0865-T100) or corresponding fluorescently labeled IgG controls.
Data collected on different days was normalized using rainbow beads (Spherotech, Lake Forest, IL; RFP-30-5A).

Zahm C.D., Colluru V, & McNeel D.G. (2017). Vaccination with High-Affinity Epitopes Impairs Antitumor Efficacy by Increasing PD-1 Expression on CD8+ T Cells. Cancer immunology research, 5(8), 630-641.

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Multiparametric Flow Cytometry Analysis

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Cell phenotypes were evaluated by multiparameter flow cytometry. Cells were incubated with a panel of labeled monoclonal antibodies (mAbs) anti-CD3 AF700, CD4 BV605, CD8 APC-H7, CD25 BV786, PD-1 APC, TIGIT BV421, and LAG3 BV711 (BD Biosciences) in staining buffer on ice in the dark for 20 min. Cells were then washed in the staining buffer and re-suspended in staining buffer and analyzed on an LSRII flow cytometer. Isotype controls were used for each experiment. Analysis of the results used FlowJo 10 software.
CFSE staining was conducted according to manufacturer’s instructions (Thermo Fisher Scientific). Briefly, cells were labeled with CFSE by adding 1 mL of freshly prepared CFSE (2 μM in PBS containing 2% EV free FCS) to cells (up to 1×10⁸ cells) in 1 mL of PBS 2% EV free FCS. The tube containing this mixture was covered with foil and incubated at 337°Cfor 5 minutes. Cells were pelleted, washed twice with 10 mL of PBS 2% EV free FCS, resuspended, counted and used for experiments.

Pando A., Fast L., Dubielecka P.M., Chorzalska A., Wen S, & Reagan J. (2021). Murine Leukemia-Derived Extracellular Vesicles Elicit Antitumor Immune Response. Journal of Blood Medicine, 12, 277-285.

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Splenocyte Activation and Phenotyping

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Splenocytes were cultured at 2 × 10⁶/mL in RPMI 1640 + l-glutamine, 10% FCS, penicillin/streptomycin (200 U/mL), 1% NaPyr, 1% HEPES, 50 µM β-MeOH, and the designated peptide (2 µg/mL). At the time points indicated, cells were stained with the following antibodies: CD3-FITC (BD 555274), CD4-BUV395 (BD 563790), CD8-BUV805 (BD 564920), LAG3-BV711 (BD 563179), PD1-PECF594 (BD 562523), TIM3-APC (eBioscience 17-5871-82), 41BB-PerCPeF710 (eBioscience 46-1371-82), CTLA4-PE (eBioscience 12-1529-42), Live/Dead Ghost dye 780 (Tonbo 13-0865-T100), or corresponding fluorescently labeled IgG controls. Cells were then fixed for 15 min at 4°C in cytofix (BD Biosciences, San Jose, CA; 554655), and frozen in FCS + 10% DMSO. After all time points were collected, cells from all times were thawed, rinsed and resuspended in PBS + 3% FCS + 1 mM EDTA and analyzed by flow cytometry. All antibodies used were at 1:100 dilutions and stained for 30 min at 4°C in a 1:4 dilution of brilliant stain buffer (BD 563794) in PBS + 3% FCS + 1 mM EDTA.

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CD8+ T Cell Activation Assay

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B cells or dendritic cells (DCs) were enriched from splenocytes of OT-1 or B6 mice inoculated with Flt3 ligand-expressing B16 tumor cells^{25 (link)} using PE-labeled antibodies specific for either CD19 or CD11c (StemCell, Seattle, WA, Cat.# 17,684) as previously described.^{26 (link)} Similarly, CD8 + T cells were isolated using a negative selection CD8 + T-cell isolation kit (StemCell, Cat. # 19,853). After enrichment, each APC subset, and a subset of purified T cells, were cultured as described above with 2 µg/mL SIINFELK or the HLA-A2 sequence from SSX2 (RLQGISPKI) as a nonspecific control peptide. Naïve OT-1 T cells were added to each cell type at a 1:1 ratio and incubated for three days, after which cells were stained and analyzed by flow cytometry with the following panel: CD3-FITC (BD 555,274), CD4-BUV395 (BD 563,790), CD8-BUV805 (BD 564,920), LAG-3-BV711 (BD 563,179), PD1-PECF594 (BD 562,523), TIM3–APC (eBioscience 17-5871-82), CTLA4–PECy7 (Tonbo 60-1522-U100), 41BB–PerCPeF710 (eBioscience 46–1371–82), and Live/Dead Ghost dye 780 (Tonbo, San Diego, CA 13–0865–T100).

Zahm C.D., Moseman J.E., Delmastro L.E, & G. Mcneel D. (2021). PD-1 and LAG-3 blockade improve anti-tumor vaccine efficacy. Oncoimmunology, 10(1), 1912892.

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Phenotyping Antigen-Specific CD8 T Cells

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Splenocytes were collected from naive OT-1 or immunized HHDII-DR1 mice as described above, cultured with 2 µg/mL (unless otherwise indicated) native SSX2-p103, SIINFEKL APL, a non-specific peptide (negative control), or phorbol 12-myristate 13-acetate (40 ng/mL, PMA, Sigma-Aldrich, St. Louis, MO; P8139) and ionomycin (2.6 µg/mL, Fisher Scientific, Waltham, MA; ICN15507001) as a positive control. After two hours golgistop (0.67µL/mL, BD 554724) was added. Cells were incubated for six additional hours (8 hours total), after which intracellular cytokine staining was performed as per the manufacturer’s protocol (Cytofix/Cytoperm Kit, BD 554714). Antibodies used for cells surface staining were: CD3-FITC (BD 555274), CD4-BUV395 (BD 563790), CD8-BUV805 (BD 564920), LAG3-BV711(BD 563179), PD1-PECF594 (BD 562523), 41BB-PerCPeF710 (eBioscience 46-1371-82). Antibodies used for intracellular staining were: TNFα-PECy7 (BD 557644), IL2-APC (eBioscience 17-7021-82), IFNγ-PE (BD 554412), and Live/Dead Ghost dye 780 (Tonbo 13-0865-T100) or corresponding fluorescently labeled IgG controls. The number of antigen-specific Th1 cells (expressing IL2 and/or TNFα and/or IFNγ) was determined as a percentage of total CD8 T cells via an “OR” Boolean gate (FlowJo software v10.1).

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