Lag 3 percp cy5, by BioLegend - Product details

1

Multiparametric T cell analysis

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T cells were re-stimulated with phorbol myristate acetate (Sigma, 50 ng/mL) and ionomycin (Sigma, 500 ng/mL). 90 minutes later, cells were treated with Brefeldin A to block cytokine secretion. 3 h later, cells were stained for surface markers and simultaneously labelled with Live/Dead Blue Viability Dye (Thermo Fisher) for 20 minutes at 4°C. Cells were washed twice and fixed overnight using a FoxP3 Fixation/Permeabilization kit (Thermo Fisher). The following day, cells were washed and stained for intracellular cytokines at room temperature for 1 h. They were then washed 3 times and analysed using an LSR Fortessa machine (Beckman Dickinson). Analysis of mean fluorescence intensity was performed using FlowJo v10.0. All experiments were performed at least two independent times. Antibodies used (at 1:100 unless otherwise noted) were TNF-PE (BioLegend, MP6-XT22, 506306), PD-1-PECy7 (BioLegend, RMP1–30, 109110) IFN-γ-FITC (BioLegend, XMG1.2, 505806), CD4-BV711 (BioLegend, RM4–5, 100550), CD8α-BV786 (BioLegend, 53–6.7, 100750), Tox-PE (Miltenyi, REA473, 130–120-716, 1:50), Tcf7-Alexa647 (Cell Signaling, C63D9, 6709), PD-L1-APC (BioLegend, 10F.9G2, 124312, 1:5000), LAG-3-PerCP-Cy5.5 (BioLegend, C9B7W, 125211), TIM-3-PE-Dazzle594 (BioLegend, B8.2C12, 134013).

Vardhana S.A., Hwee M.A., Berisa M., Wells D.K., Yost K.E., King B., Smith M., Herrera P.S., Chang H.Y., Satpathy A.T., van den Brink M.R., Cross J.R, & Thompson C.B. (2020). Impaired mitochondrial oxidative phosphorylation limits the self-renewal of T cells exposed to persistent antigen. Nature immunology, 21(9), 1022-1033.

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2

Multiparametric Flow Cytometry Staining

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Staining for surface markers was performed by incubating cells with antibody at 1:200 dilutions in fluorescence-activated cell sorting buffer (0.1% BSA in PBS) for 30 min at 4 °C. For intracellular cytokine staining of IFNγ and TNFα, surface markers were stained before fixation/permeabilization (BD Cytofix/Cytoperm Kit, #554714; BD Biosciences) (40 (link)). Flow antibodies were purchased from BioLegend with the information as follows: CD8a-BV786 (#563332), CD11c-BV786 (#563735), PD1-PE/Cy7 (#109109), TIM3-APC (#119705), KLRG1-FITC (#138409), LAG3-PerCP/Cy5.5 (#125211), TNFα-PE/Cy7 (#506323), and Granzyme B-Alexa 700 (#372221). Samples were acquired on BD LSRFortessa flow cytometer and analyzed with FlowJo software (https://www.flowjo.com/solutions/flowjo) (Tree Star) unless otherwise stated.

Zhang J., Ye Z.W., Bräutigam L., Chakraborty P., Luo Z., Culpepper J., Aslam M., Zhang L., Johansson K., Haeggström J.Z., Xu J., Olsson M., Townsend D.M., Mehrotra S., Morgenstern R, & Tew K.D. (2023). A role for microsomal glutathione transferase 1 in melanin biosynthesis and melanoma progression. The Journal of Biological Chemistry, 299(8), 104920.

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3

Flow Cytometric Analysis of CAR-T Cell Phenotype

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Surface protein expression was determined by flow cytometry. After electroporation for 5 days, 1×10⁶ cells were incubated with APC-CD4, PE/Cy7-TCR (or PE-TCR) and FITC-CD3 antibodies (Biolegend) for 30 mins. For the CD22BBz CAR transduced T cells were incubated with 0.2 μg CD22-Fc (R&D system) in 100 μL PBS for 30 mins, and then stained with PE-IgG-Fc (Biolegend). For the CD19BBz CAR detection, the transduced T cells were stained with APC-anti-DYKDDDDK Tag (Biolegend). Stained cells were measured and sorted on BD FACSAria II. For the T cell exhaustion assay, T cells from various groups were co-cultured with NALM6 cells at 0.5:1 E:T ratio for 24 hours. 1×10⁶ cells were incubated with 0.2 μg CD22-Fc (R&D Systems) in 100 μL PBS for 30 mins and then stained with PE-IgG-Fc, PD-1-FITC, TIGIT-APC and LAG3-Percp/cy5.5 (Biolegend) for 30 mins. After washing twice, the stained cells were measured and sorted on BD FACSAria II, and analyzed using FlowJo software version 9.9.4 or 10.3 (Treestar, Ashland, OR).

Dai X., Park J.J., Du Y., Kim H.R., Wang G., Errami Y, & Chen S. (2019). One-step generation of modular CAR-T with AAV-Cpf1. Nature methods, 16(3), 247-254.

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4

Multiparametric T cell analysis

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T cells were re-stimulated with phorbol myristate acetate (Sigma, 50 ng/mL) and ionomycin (Sigma, 500 ng/mL). 90 minutes later, cells were treated with Brefeldin A to block cytokine secretion. 3 h later, cells were stained for surface markers and simultaneously labelled with Live/Dead Blue Viability Dye (Thermo Fisher) for 20 minutes at 4°C. Cells were washed twice and fixed overnight using a FoxP3 Fixation/Permeabilization kit (Thermo Fisher). The following day, cells were washed and stained for intracellular cytokines at room temperature for 1 h. They were then washed 3 times and analysed using an LSR Fortessa machine (Beckman Dickinson). Analysis of mean fluorescence intensity was performed using FlowJo v10.0. All experiments were performed at least two independent times. Antibodies used (at 1:100 unless otherwise noted) were TNF-PE (BioLegend, MP6-XT22, 506306), PD-1-PECy7 (BioLegend, RMP1–30, 109110) IFN-γ-FITC (BioLegend, XMG1.2, 505806), CD4-BV711 (BioLegend, RM4–5, 100550), CD8α-BV786 (BioLegend, 53–6.7, 100750), Tox-PE (Miltenyi, REA473, 130–120-716, 1:50), Tcf7-Alexa647 (Cell Signaling, C63D9, 6709), PD-L1-APC (BioLegend, 10F.9G2, 124312, 1:5000), LAG-3-PerCP-Cy5.5 (BioLegend, C9B7W, 125211), TIM-3-PE-Dazzle594 (BioLegend, B8.2C12, 134013).

Vardhana S.A., Hwee M.A., Berisa M., Wells D.K., Yost K.E., King B., Smith M., Herrera P.S., Chang H.Y., Satpathy A.T., van den Brink M.R., Cross J.R, & Thompson C.B. (2020). Impaired mitochondrial oxidative phosphorylation limits the self-renewal of T cells exposed to persistent antigen. Nature immunology, 21(9), 1022-1033.

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5

Flow Cytometric Analysis of CAR-T Cell Phenotype

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Surface protein expression was determined by flow cytometry. After electroporation for 5 days, 1×10⁶ cells were incubated with APC-CD4, PE/Cy7-TCR (or PE-TCR) and FITC-CD3 antibodies (Biolegend) for 30 mins. For the CD22BBz CAR transduced T cells were incubated with 0.2 μg CD22-Fc (R&D system) in 100 μL PBS for 30 mins, and then stained with PE-IgG-Fc (Biolegend). For the CD19BBz CAR detection, the transduced T cells were stained with APC-anti-DYKDDDDK Tag (Biolegend). Stained cells were measured and sorted on BD FACSAria II. For the T cell exhaustion assay, T cells from various groups were co-cultured with NALM6 cells at 0.5:1 E:T ratio for 24 hours. 1×10⁶ cells were incubated with 0.2 μg CD22-Fc (R&D Systems) in 100 μL PBS for 30 mins and then stained with PE-IgG-Fc, PD-1-FITC, TIGIT-APC and LAG3-Percp/cy5.5 (Biolegend) for 30 mins. After washing twice, the stained cells were measured and sorted on BD FACSAria II, and analyzed using FlowJo software version 9.9.4 or 10.3 (Treestar, Ashland, OR).

Dai X., Park J.J., Du Y., Kim H.R., Wang G., Errami Y, & Chen S. (2019). One-step generation of modular CAR-T with AAV-Cpf1. Nature methods, 16(3), 247-254.

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Lag 3 percp cy5

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5 protocols using lag 3 percp cy5

Multiparametric T cell analysis

Multiparametric Flow Cytometry Staining

Flow Cytometric Analysis of CAR-T Cell Phenotype

Multiparametric T cell analysis

Flow Cytometric Analysis of CAR-T Cell Phenotype

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