Isolated CD8+ T cells from PBMCs (see above) were activated in 96-well plates coated with 5mg/ml plate bound Ultra-LEAF™ anti-CD3 Ab (OKT3, Biolegend) and 2mg/ml plate bound Ultra-LEAF™ anti-CD28 Ab (CD28.2, Biolegend) at a density of 1 × 105 cells per well, in a 1:1 mix of Aim 5 (Gibco) and RPMI medium (Gibco), supplemented with 10% AB serum (Life Technologies), 100 U/ml penicillin (Roche), 100 mg/ml streptomycin (Roche). After 2 h, cells were either left untreated or were treated with 5 nM LCKi inhibitor (Merck Milipore) for the indicated time periods. Cells were subsequently washed 3 times with medium to remove already secreted IFNγ, and cells were then cultured for 3h in 40 ml fresh control medium or fresh medium containing 5 nM LCK inhibitor. Subsequently, supernatants were collected and IFNγ concentrations were determined using the BD™ Cytometric Bead Array (CBA) Human Th1/Th2/Th17 Cytokine Kit, according to the manufacturer’s protocol.
Ultra leaf anti cd3 ab okt3
Manufactured by BioLegend
Ultra-LEAF™ anti-CD3 Ab (OKT3) is a mouse monoclonal antibody that binds to the CD3 epsilon chain of the T cell receptor complex. It is designed for research use.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi medium, by Thermo Fisher Scientific (2 mentions)
Aim 5, by Thermo Fisher Scientific (2 mentions)
Cytometric bead array human th1 th2 th17 cytokine kit, by BD (2 mentions)
Ab serum, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Roche (2 mentions)
Ultra leaf anti cd28 ab cd28, by BioLegend (2 mentions)
Lcki inhibitor, by Merck Group (2 mentions)
Penicillin, by Roche (2 mentions)
2 protocols using ultra leaf anti cd3 ab okt3
1
CD8+ T cell activation and IFNγ analysis
2
CD8+ T cell activation and IFNγ analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Isolated CD8+ T cells from PBMCs (see above) were activated in 96-well plates coated with 5mg/ml plate bound Ultra-LEAF™ anti-CD3 Ab (OKT3, Biolegend) and 2mg/ml plate bound Ultra-LEAF™ anti-CD28 Ab (CD28.2, Biolegend) at a density of 1 × 105 cells per well, in a 1:1 mix of Aim 5 (Gibco) and RPMI medium (Gibco), supplemented with 10% AB serum (Life Technologies), 100 U/ml penicillin (Roche), 100 mg/ml streptomycin (Roche). After 2 h, cells were either left untreated or were treated with 5 nM LCKi inhibitor (Merck Milipore) for the indicated time periods. Cells were subsequently washed 3 times with medium to remove already secreted IFNγ, and cells were then cultured for 3h in 40 ml fresh control medium or fresh medium containing 5 nM LCK inhibitor. Subsequently, supernatants were collected and IFNγ concentrations were determined using the BD™ Cytometric Bead Array (CBA) Human Th1/Th2/Th17 Cytokine Kit, according to the manufacturer’s protocol.
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