NK cell degranulation was measured as described previously 26 (link). Briefly, PBMCs were incubated with K562 cells [5:1 effector : target (E : T) ratio] for 3 h at 37°C following overnight stimulation with a combination of 50 ng/ml recombinant human (rh)IL-12 and rhIL-18 (Miltenyi Biotec, R&D Systems). CD107a-PE antibody (BD Biosciences) was added at the time of stimulation with target cells along with 1 mM monensin prior to staining and acquisition.
Cd107a pe antibody
Manufactured by BD
Sourced in United States
The CD107a-PE antibody is a laboratory reagent used in flow cytometry applications. It is a monoclonal antibody conjugated with the fluorescent dye Phycoerythrin (PE), which binds to the CD107a cell surface antigen. This antibody can be used to detect and analyze cells expressing the CD107a protein.
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5 protocols using cd107a pe antibody
1
NK Cell Degranulation Measurement
2
Intracellular Cytokine Staining of Peptide-Stimulated PBMCs
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PBMCs were in vitro stimulated with peptide as described previously. At 10–14 days post simulation, the cells were used for ICS. Cells were incubated with peptide or a no-peptide control and a CD107a-PE antibody (BD) for 1 hour, followed by addition of GolgiPlug (BD). After a 4-hour incubation period, the cells were washed and stained with antibodies for extracellular markers: CD3–PC/H7, CD4–FITC, and CD8–PerCP. Dead cells were stained with FVS-510. Next, the cells were permeabilized using fixation/permeabilization buffer (Invitrogen), followed by staining with intracellular antibodies: IFN-γ–APC and tumor necrosis factor alpha (TNF-α)–BV421. Data were acquired using a BD Canto II flow cytometer and analyzed using FlowJo. The gating strategy is presented in online supplemental figure S6 , and the antibodies used in this study are listed in online supplemental table S2 .
3
Quantifying NK Cell Degranulation via CD107a Expression
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CD107a expression on NK cells was measured to analyze NK cell degranulation. Peripheral blood mononuclear cells stimulated overnight with 10 ng/ml IL-12 and 50 ng/ml IL-18 or untreated PBMCs were washed and treated afterward either with K562 cells with an effector-to-target (E:T) ratio of 15:1 or without target cells for 4 h. Peripheral blood mononuclear cells stimulated only with PMA (50 ng/ml) plus ionomycin (1 μg/ml) served as positive controls. Prior to 4 h incubation with the CD107a-PE antibody (BD Biosciences, San Diego, CA, United States) monensin (5 μg/ml, BioLegend, Coblenz, Germany) and brefeldin A (5 μg/ml) were added. For every condition, CD107a-unstained controls were included as well as unstimulated samples with CD107a staining to detect spontaneous degranulation. After 4 h of incubation, samples were washed and extracellular backbone immunofluorescence staining was performed as described above (see section “Surface Staining and Determination of Extracellular Markers”).
4
NK Cell Degranulation Assay Protocol
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Degranulation assays were performed as previously described [48 (link)]. Briefly, activated NK cells and target cells were mixed at a 1:1 ratio in the presence of the CD107a-PE antibody (BD Biosciences, San Jose, CA, USA) and incubated for 1 h at 37°C. GolgiStop (6 μg/mL; BD Biosciences, San Jose, CA, USA) was then added, and cells were incubated for an additional 4 h at 37°C. When required, NB target cells were first incubated with ch14.18 anti-GD2 mAb (1 μg/mL) before the co-culture with NK cells. After staining with CD56-APC antibody and addition of 7-AAD, surface expression of CD107a was assessed using a Fortessa cytometer (BD Biosciences), and data analysis was performed using the FlowJo program (Tree Star).
5
CD107a Degranulation Assay for T Cells
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To assess CD107a degranulation, we plated 1 × 105 T cells and 5 × 105 stimulator target cells per well in round-bottom 96-well plates, to a final volume of 200 μl in complete R10 medium, in triplicates. The CD107a-PE antibody (BD) was added into each well and incubated at 37°C for 4 h, along with surface staining for CD8 (BioLegend, San Diego, CA, USA) and CD3 and then analyzed by flow cytometry.
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