MAIT cell functions were determined in vitro using a formaldehyde-fixed E. coli stimulation (D21 strain, fixed in 1% formaldehyde for 5 min multiplicity of infection=5) in the presence of 1.25 μg/ml anti-CD28 mAb (clone L293, BD Biosciences) and, in selected experiments, 20 μg/ml anti-MR1 mAb (clone 26.5; Biolegend) or IgG2 isotype control (clone MOPC-173, BioLegend). PBMCs or FGT-derived MNCs were further cultured for 24 hours at 37°C and 5% CO2 in RPMI medium supplemented with 10% fetal calf serum and 50 μg/ml gentamicin (Gibco). monensin (Golgi Stop, BD Biosciences) was added during the last 6 hours of the stimulation. Cells stimulated for 6 h with PMA/ionomycin (Leukocyte Activation Cocktail, BD Biosciences) and in the presence of monensin were included in parallel as positive controls.
Pma ionomycin leukocyte activation cocktail
Manufactured by BD
PMA/ionomycin (Leukocyte Activation Cocktail) is a laboratory reagent used to stimulate the activation of leukocytes, such as T cells and natural killer cells. It is a combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin, which together trigger intracellular signaling pathways that lead to the activation and proliferation of these immune cells.
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Lab products found in correlation
2 protocols using pma ionomycin leukocyte activation cocktail
1
MAIT Cell Functional Assay with E. coli
2
MAIT Cell Functional Assay with E. coli
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MAIT cell functions were determined in vitro using a formaldehyde-fixed E. coli stimulation (D21 strain, fixed in 1% formaldehyde for 5 min multiplicity of infection=5) in the presence of 1.25 μg/ml anti-CD28 mAb (clone L293, BD Biosciences) and, in selected experiments, 20 μg/ml anti-MR1 mAb (clone 26.5; Biolegend) or IgG2 isotype control (clone MOPC-173, BioLegend). PBMCs or FGT-derived MNCs were further cultured for 24 hours at 37°C and 5% CO2 in RPMI medium supplemented with 10% fetal calf serum and 50 μg/ml gentamicin (Gibco). monensin (Golgi Stop, BD Biosciences) was added during the last 6 hours of the stimulation. Cells stimulated for 6 h with PMA/ionomycin (Leukocyte Activation Cocktail, BD Biosciences) and in the presence of monensin were included in parallel as positive controls.
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