BMDCs were seeded at 1 × 106 cells per well in 6 wells plate (CELLTER, China). CD4+ T cells from OT-II mice or CD8+ T cells from OT-I mice were purified by CD4+ or CD8+ microbeads (Miltenyi Biotec, Germany). To detect cell proliferation, cells were labeled with CFSE (2 μM; Selleckchem, Houston, USA) at 37 °C for 20 min. To detect cell activation, cells were resuspended with RPMI-1640 containing 10% FBS and IL-2 (20 ng/ml) and seeded at 5 × 104 cells per well in round-bottomed 96-well plates. The next day, BMDCs were incubated with OVA (1 mg/ml; Bohu Biotec, Shanghai, China) and OVA323–339 (1 μg/ml; Sangon Biotech, Shanghai, China) or OVA257–264 (1 μg/ml; Sangon Biotech, Shanghai, China) for 6 h, washed twice with RPMI-1640 containing 10% FBS and IL-2 (20 ng/ml; Pepro Tech, USA) and co-cultured with purified T cells at a ratio of DC: T cell = 1: 10 for 24 h, flow cytometry analysis of the expression of activation index CD69. For 72 h, proliferation was determined by detecting the fluorescence intensity of CFSE via flow cytometry.
Carboxyfluorescein succinimidyl ester (cfse)
Manufactured by Selleck Chemicals
Sourced in United States
CFSE is a fluorescent dye used for labeling and tracking cells. It binds to intracellular proteins, allowing the visualization and monitoring of cell division and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Ova323 339, by Sangon (1 mentions)
Ova257 264, by Sangon (1 mentions)
Cd4 or cd8 microbeads, by Miltenyi Biotec (1 mentions)
Isotype control antibody, by BioLegend (1 mentions)
Antihuman pd l1 neutralizing antibody, by BioLegend (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Lsrii ow cytometer, by BD (1 mentions)
Cd8 antibody, by BioLegend (1 mentions)
Cellquest software, by BD (1 mentions)
4 protocols using carboxyfluorescein succinimidyl ester (cfse)
1
Co-culture of BMDCs and T Cells for Activation and Proliferation
2
Autologous T Cell Activation and Proliferation
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Autologous T cells were labeled with 5 µM carboxyfluorescein succinimidyl ester (CFSE, Selleck Chemicals) for 25 min at 4°C and preactivated with plate-bound 2 µg/mL anti-CD3 (Thermo Fisher) at 37°C, 5% CO2 for 16–18 hours. Three days after activation, the T cells were incubated with the macrophages for 2 days at a T cell:macrophage ratio of 5:1. The proliferation of T cells was measured by CFSE staining and flow cytometry. In some experiments, 5 mg/mL antihuman PD-L1 neutralizing antibody (Biolegend), 5 mg/mL isotype control antibody (Biolegend) or 4 mg/mL 1-MT (Selleck Chemicals) was added in the coculture.
3
CFSE-Based Proliferation Assay for γδ T Cells
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4
CD8+ T Cell Proliferation Assay
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To analyze the CD8 + T cell among CD45 + cells from the of mice, CD8 + T cells were collected and concentrated, and they were incubated with 5 µL of speci c uorescent CD8 antibody (100705, Biolegend, USA) and speci c uorescent CD45 antibody (368507, Biolegend) for 20 min. Saline (500 µL) was added separately into the cell solution, which was followed by centrifugation at 5000 rpm for 5 min.
The supernatant was removed, and the collected cells at the bottom were dispersed into 300 µL of saline.
Finally, the effective separation rate was analyzed by counting 1.2 × 10 4 cells per sample using an LSR II ow cytometer (BD Biosciences, CA, USA) and Cell Quest software (BD Biosciences).
CD8 + T cell proliferation assay CD8 T + cell was puri ed from peripheral blood mononuclear cells (PBMCs) of healthy female volunteers. Cell purity was checked. CD8 + T cellwas labeled with 1μM CFSE (S8269, selleck, USA) and stimulated with CD3/CD28 antibody and co-cultured with vector transfected, LC3plasmid, and LC3 + NLRC5 HEC-1A cells, in 96-well plates at a ratio of 5: 1 for 3 days. CD8 + T cell was collected, and the dilution of intracellular CFSE caused by proliferation was calculated using ow cytometry.
The supernatant was removed, and the collected cells at the bottom were dispersed into 300 µL of saline.
Finally, the effective separation rate was analyzed by counting 1.2 × 10 4 cells per sample using an LSR II ow cytometer (BD Biosciences, CA, USA) and Cell Quest software (BD Biosciences).
CD8 + T cell proliferation assay CD8 T + cell was puri ed from peripheral blood mononuclear cells (PBMCs) of healthy female volunteers. Cell purity was checked. CD8 + T cellwas labeled with 1μM CFSE (S8269, selleck, USA) and stimulated with CD3/CD28 antibody and co-cultured with vector transfected, LC3plasmid, and LC3 + NLRC5 HEC-1A cells, in 96-well plates at a ratio of 5: 1 for 3 days. CD8 + T cell was collected, and the dilution of intracellular CFSE caused by proliferation was calculated using ow cytometry.
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