Human peripheral blood mononuclear cells (PBMC) were isolated from the blood of 14 healthy donors using Hypaque-Ficoll (GE Healthcare, Amersham, UK) density centrifugation, with informed consent and full ethical approval (REC ref 06/Q1701/120). 3 × 105 PBMCs were stimulated overnight with 1 ng/mL recombinant human IL-15. Peptide pulsed 721.174 targets were prepared as for the stabilization assays. Target cells were resuspended with PBMCs at an effector-to-target (E:T) ratio of 5:1 in fresh R10 medium containing peptide and anti-CD107a-Alexa-fluor-647 antibody (eBioscience, Hatfield, UK). Cells were incubated for 1 h at 26°C, then 6 μg/mL Golgi-Stop™ (BD Biosciences) was added, and incubated for a further 3 h at 26°C. Cells were washed in wash buffer (1% BSA/0.1% NaN3) and blocked with blocking buffer (10% human serum in PBS) for 30 min and then stained with the following antibodies: anti-CD3-PerCP (Biolegend, San Diego, US), anti-human CD56-PE and anti-human CD158b-FITC (both BD Biosciences). Cells were fixed in 1% PFA and analyzed by flow cytometry. Individual assays for each donor were performed once in duplicate and the mean value used for subsequent analysis.
Anti human cd158b fitc
Manufactured by BD
Sourced in United States
Anti-human CD158b-FITC is a fluorescently-labeled monoclonal antibody that binds to the CD158b receptor expressed on certain immune cells. This product is intended for research use only.
Automatically generated - may contain errors
2 protocols using anti human cd158b fitc
1
Quantifying NK cell activation by IL-15
2
Cytotoxicity Assay of Human PBMC Stimulated with IL-15
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Human PBMC were isolated from the blood of 14 healthy donors using Hypaque‐Ficoll (GE Healthcare, Amersham, UK) density centrifugation, with informed consent and full ethical approval (REC ref 06/Q1701/120). A total of 3 × 105 PBMCs were stimulated overnight with 1 ng/mL recombinant human IL‐15. Peptide pulsed 721.174 targets were prepared as for the stabilization assays. Target cells were resuspended with PBMCs at an effector‐to‐target (E:T) ratio of 5:1 in fresh R10 medium containing peptide and anti‐CD107a‐Alexa‐fluor‐647 antibody (eBioscience, Hatfield, UK). Cells were incubated for 1 h at 26°C, then 6 μg/mL Golgi‐StopTM (BD Biosciences) was added, and incubated for a further 3 h at 26°C. Cells were washed in wash buffer (1% BSA/0.1% NaN3) and blocked with blocking buffer (10% human serum in PBS) for 30 min and then stained with the following antibodies: anti‐CD3‐PerCP (Biolegend, San Diego, USA), anti‐human CD56‐PE, and anti‐human CD158b‐FITC (both BD Biosciences). Cells were fixed in 1% PFA and analyzed by flow cytometry. Individual assays for each donor were performed once in duplicate and the mean value used for subsequent analysis.
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