After being thawed, 1x106 PBMCs in completed medium were washed with PBS-0.5% fetal calf serum and stained with the fluorescently labeled anti-human antibodies CD4-FITC (BD Biosciences Cat#555346), PD1-PE (BD Biosciences Cat#557946), LAG3-BV421 (BD Biosciences Cat#565720), TIM3-AlexaFluor641 (BD Biosciences Cat#565558) and their corresponding isotype controls; FITC Mouse IgG1,k Isotype (BD Biosciences Cat#551954); PE Mouse IgG1,k Isotype (Beckman Coulter Cat#A07796), BV421 Mouse IgG1,k Isotype (BD Biosciences Cat#562438) and Alexa Fluor 647 Mouse IgG1 Isotype (BD Biosciences Cat#565571) at 4°C, for 30 min in the dark. For the identification and quantitation of exhausted CD4+ T cell subsets, 100,000 events were acquired per sample and analyzed in a Fortessa equipped with the FACS DIVA software (BD Biosciences, San Jose, CA). BD FACS Express software (BD Biosciences, San Jose, CA) was used for data analysis. Results were expressed as a percentage of positive cells.
Lag3 bv421
Manufactured by BD
LAG3-BV421 is a fluorescently-labeled monoclonal antibody that binds to the Lymphocyte Activation Gene 3 (LAG3) receptor. LAG3 is an immune checkpoint molecule expressed on activated T cells, regulatory T cells, and natural killer cells. The BV421 fluorescent dye is used for flow cytometry analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pd 1 pe, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Cd4 fitc, by BD (1 mentions)
Pd 1 apc, by BD (1 mentions)
Ctla4 bv786, by BD (1 mentions)
Tim 3 bv650, by BD (1 mentions)
Pd l1 apc, by BD (1 mentions)
Cd3 percp cy5, by BD (1 mentions)
Lymphocyte separation medium, by MP Biomedicals (1 mentions)
Cd8 pe cy7, by BD (1 mentions)
Facscelesta, by BD (1 mentions)
Cd45 apc cy7, by BD (1 mentions)
2 protocols using lag3 bv421
1
Quantification of Exhausted CD4+ T Cells
2
Immune Checkpoint Modulation in Liver Cancer
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Peripheral blood mononuclear cells (PBMC) were extracted from healthy volunteers by density gradient centrifugation in a lymphocyte separation medium (MP Biomedicals, Irvine, CA, USA). T cells in PBMC were activated and expanded with CD3, CD28 antibody, and 10 ng/ml IL-2 (Thermo Fisher Scienti c, Waltham, MA, USA), and then co-cultured with Hep3B cells at a ratio of 10:1 in the presence of a uorescent caspase 3 substrate (BD Biosciences, San Jose, CA, USA).
After 48h, PBMC and Hep3B were collected separately. The expression of immune checkpoints and apoptosis rate were measured by ow cytometry (BD FACSCelesta). All antibodies were purchased from BD Biosciences, including CD45-APC-CY7, CD3-PERCP-CY5.5, CD8-PE-CY7, PD1-APC, TIM3-BV650, LAG3-BV421, CTLA4-BV786, Caspase3-PE, and PDL1-APC antibodies.
After 48h, PBMC and Hep3B were collected separately. The expression of immune checkpoints and apoptosis rate were measured by ow cytometry (BD FACSCelesta). All antibodies were purchased from BD Biosciences, including CD45-APC-CY7, CD3-PERCP-CY5.5, CD8-PE-CY7, PD1-APC, TIM3-BV650, LAG3-BV421, CTLA4-BV786, Caspase3-PE, and PDL1-APC antibodies.
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