Lag3 apc

Manufactured by BioLegend

LAG3-APC is a fluorescently-labeled antibody that binds to the LAG3 (Lymphocyte-Activation Gene 3) protein. LAG3 is a cell surface receptor that is expressed on various immune cells, including T cells and natural killer cells. The APC (Allophycocyanin) fluorescent label allows for the detection and analysis of LAG3-expressing cells using flow cytometry techniques.

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5 protocols using lag3 apc

Isolation and Analysis of Tumor-Associated Immune Cells

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At day 21 post tumor implantation, mice were sacrificed using a lethal dose of ketamine/xylazine cocktail. The brain and cervical lymph nodes (LN) were harvested and passed through a 40-μm strainer. A 30–37–60% Percoll gradient (GE Healthcare, Buck-inghamshire, UK) was used to isolate immune cell populations from brain tumors and the draining lymph nodes (LNs). After centrifugation, the 37–60% interface contained lymphocytes, monocytes and microglia in the case of brain tumors, and contained lymphocytes and monocytes in the case of draining LNs.
For flow cytometric analysis, lymphocytes were stained with CD8 PerCp-Cy5.5 Clone: 53–6.7 (eBioscience), CD3 FITC Clone: 17A2 (eBioscience), CD4 APCH7 Clone: GK1.5 (BD Biosciences), FoxP3 PE Clone: NNRF-30 (eBioscience), IFNγ BV421 Clone: XMG 1.2 (Biolegend), LAG-3 APC Clone: C9B7W, PD-1 PE-Cy7 Clone: J43 and fixable aqua L/D stain (Life Technologies). Appropriate isotype controls were used.
All flow cytometry experiments were performed on a LSRII (BD Biosciences) and analysis was performed using FlowJo software (TreeStar, Ashland, OR).

Harris-Bookman S., Mathios D., Martin A.M., Xia Y., Kim E., Xu H., Belcaid Z., Polanczyk M., Barberi T., Theodros D., Kim J., Taube J.M., Burger P.C., Selby M., Taitt C., Korman A., Ye X., Drake C.G., Brem H., Pardoll D.M, & Lim M. (2018). Expression of LAG-3 and efficacy of combination treatment with anti-LAG-3 and anti-PD-1 monoclonal antibodies in glioblastoma. International journal of cancer, 143(12), 3201-3208.

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Multicolor Flow Cytometry of PBMC

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We isolated peripheral blood mononuclear cells (PBMC) from peripheral blood by Ficoll gradient and we stored them in liquid nitrogen for batched analysis. For multicolor flow cytometry analyses, we used the following monoclonal antibodies: from Becton Dickinson (BD, Franklin Lakes, NJ), CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CD71-PE, IgD-PerCP-Cy5.5, CD25-APC-Cy7, CD138-BV421, CCR4-PE, CD27-PE, CD95-BV421, CD28-BV421, TIM3-BV421, CCR6-BV421, CCR7-AF700, CXCR3-PE, IL-2-PE, IL-17-BV786, and IFN-γ-PE-Cy7; from Biolegend (San Diego, CA), CD19-BV510, CD56-FITC, CD27-APC, CD127-FITC, CD57-PerCp-Cy5.5, PD-1-APC-Cy7, CXCR5-FITC, LAG3-APC, IL-10-PerCP-Cy5.5, and TNF-α-FITC. From eBioscience (San Diego, CA), CD4-PE-Cy7, CD21-PE, and CD24-APC-Cy7 were used. From Miltenyi Biotec (Auburn, CA), CD25-APC and anti-KLRG1-PE were used. From Beckman Coulter (Indianapolis, IN), CD38-PE-Cy7 was used.
We performed intracellular staining for IL-2, IL-4, IL-17, IFN-g, and TNF-a together with extracellular markers for CD4⁺, CD8⁺, and CD19⁺. Cells were fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions.
Data were acquired (> 1 × 10⁶ events) on a 3-laser FACSLyric flow cytometer (BD Biosciences) and analyzed with FlowJo^® software.

Tawhari I., Hallak P., Bin S., Yamani F., Safar-Boueri M., Irshad A., Leventhal J., Ansari M.J., Cravedi P, & Gallon L. (2022). Early calcineurin-inhibitor to belatacept conversion in steroid-free kidney transplant recipients. Frontiers in Immunology, 13, 1096881.

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Engineered CAR T Cell Generation

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PBMC were isolated from whole peripheral blood, acquired from healthy volunteer donors at the University Children’s Hospital Tuebingen, by Ficoll-Paque density gradient centrifugation (Biocoll, Biochrom). T cells were sequentially isolated using CD4 and CD8 microbeads (Miltenyi Biotec) and mixed at a 1:1 ratio. T cells were activated with TransAct^TM (anti-CD3 and anti-CD28 agonistic signal, Miltenyi Biotec) and cultivated in TexMACS media (Miltenyi Biotec) supplemented with 10 ng/mL IL7 and 5 ng/mL IL15 (Miltenyi Biotec). After 24 h, activated T cells were transduced at a multiplicity of infection (MOI) of 3. Transduced T cells were maintained at 0.5–2 × 10⁶ cells/mL in IL7/IL15 containing TexMACS® media. On day +7, CAR transduction efficiency and CD4/CD8 ratio were determined by flow cytometry using the antibodies: CD4-BUV395 (SK3, BD Bioscience), CD8-APC (BW135/80, Miltenyi Biotec) and EGFR-PE (AY13, BioLegend). Tonic signaling was assessed by expression of the exhaustion markers PD-1, TIM-3, and LAG-3 via flow cytometry on day +21 of culturing using the antibodies: EGFR-FITC (13/EGFR (RUO), BD Bioscience); PD-1-PE (PD1.3.1.3, Miltenyi); TIM-3-PE-/Dazzle594 (F38-2E2, BioLegend); LAG-3-APC (7H2C65, BioLegend) and LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (ThermoFisher).

Seitz C.M., Schroeder S., Knopf P., Krahl A.C., Hau J., Schleicher S., Martella M., Quintanilla-Martinez L., Kneilling M., Pichler B., Lang P., Atar D., Schilbach K., Handgretinger R, & Schlegel P. (2019). GD2-targeted chimeric antigen receptor T cells prevent metastasis formation by elimination of breast cancer stem-like cells. Oncoimmunology, 9(1), 1683345.

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Comprehensive CAR and Immunophenotyping Analysis

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FACS analysis of cell surface CAR and protein expression was performed using an LSR II Fortessa flow cytometer (BD Biosciences). CD22-CAR was detected by incubation with 22-Fc (R&D Systems), followed by incubation with human IgG-specific PE-F(ab)2 (Thermo Fisher Scientific). The following human monoclonal antibodies were used for detection of cell surface proteins: CD22-APC, CD22-PE, CD19-Pacific Blue, CD45-PerCP/Cy5.5, CD3-APC/Cy7, PD1-PE/Cy7, LAG3-APC, TIM3-Pacific Blue, CD8-APC, CD8-PE/Cy7, CD45RA-APC, CD45RO-PE/Cy7, CCR7-Pacific Blue, CD4-Pacific Blue, CD69-APC (all from BioLegend). CD22 site density was determined using QuantiBrite-PE beads (BD Biosciences) using methods previously described (PMID: 20872890). Dead cells were identified using eFluor 506 fixable viability dye (Thermo Fisher Scientific). GFP-expressing leukemia was identified through the FITC channel.

Ramakrishna S., Highfill S.L., Walsh Z., Nguyen S.M., Lei H., Shern J.F., Qin H., Kraft I., Stetler-Stevenson M., Yuan C.M., Hwang J., Feng Y., Zhu Z., Dimitrov D., Shah N.N, & Fry T.J. (2019). Modulation of Target Antigen Density Improves CAR T Cell Functionality and Persistence. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(17), 5329-5341.

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Phenotypic Analysis of CAR-T Cells

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The in vivo expansion and phenotype of CAR-T was analyzed postmortem in peripheral blood of CAR-T treated mice. Red blood cells were lysed with aqua followed by 10x PBS. Remaining cells were stained with the following fluorophore conjugated antibodies: murineCD45-APC-eflour780 (30F11, eBioscience); EGFR-FITC (13/EGFR(RUO), BD Bioscience); CD25-BUV737 (2A3, BD Bioscience); PD-1-PE (PD1.3.1.3, Miltenyi); TIM-3-PE-/Dazzle594 (F38-2E2, BioLegend); LAG-3-APC (7H2C65, BioLegend) and LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (ThermoFisher). To enumerate the number of T cells (mCD45⁻hCD3⁺) and CAR-T (mCD45⁻hCD3⁺hEGFRt⁺) per microlitre of blood, all tubes were recorded exhaustively on a BD^TM LSR II flow cytometer. Gating strategy is demonstrated in Supplementary Figure 6a. CAR-T activation was determined by CD25 expression. Terminal exhaustion was defined as expression of the three inhibitory receptors PD-1, TIM-3, and LAG-3.

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