Freshly enriched splenic NK cells were incubated in vitro with 20 μg/ml purified anti-NK1.1 (PK136). Antibody-coated NK cells were stained at 4°C in HBSS+1%FCS and probenecid with the calcium indicator dye Fluo-4 (Life Technologies). Events were recorded for 30 secs in the flow cytometer, after which purified Goat-Anti-Mouse IgG (H + L) Polyclonal Antibody (Jackson Immunoresearch) (20ug/ml) was added. Measurement of real-time iCa mobilization was subsequently performed for up to 4 mins. Receptor-induced iCa mobilization was quantified by determining the Area Under the Curve (AUC) over time using the FlowJo software.
Goat anti mouse igg h l polyclonal antibody
Manufactured by Jackson ImmunoResearch
Goat-Anti-Mouse IgG (H + L) Polyclonal Antibody is a purified secondary antibody produced in goats and directed against mouse immunoglobulin G (IgG), including both heavy and light chains. This antibody is designed for use in various immunological techniques.
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3 protocols using goat anti mouse igg h l polyclonal antibody
1
Measuring NK cell calcium mobilization
2
Mouse NK Cell Phenotyping and Functional Assays
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All surface antigen staining procedures were performed for 20 min at 4°C in FACS buffer (PBS + 2% FBS). Data were acquired from an LSR Fortessa flow cytometer (BD Biosciences) and analysed using FlowJo v9.9.6 (Tree Star). The antibodies used for surface staining include CD3 (145-2C11), NKp46 (29A1.4) from BD Biosciences, and NK1.1 (PK136) from BioLegend. Antibodies used for in vitro stimulation and killing assays include NKp46 (polyclonal; R&D Systems), NK1.1 (PK136; BioLegend), CD107a (1D4B; BD Biosciences), and IFN-γ (XMG1.2; BD Biosciences). Dead cells were determined using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen). Rat anti-mouse antibodies used for mouse NK cell enrichment obtained from BioLegend include CD3 (17A2), CD19 (6D5), Ter119 (TER-119), Gr-1 (RB6.8C5), CD4 (RM4–5), and CD8 (53–6.7). The secondary antibody used to cross-link mouse NK1.1 antibodies in the calcium flux experiments is goat anti-mouse IgG (H + L) polyclonal antibody (Jackson Immunoresearch).
3
Measuring NK Cell Calcium Mobilization
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Freshly purified NK cells from DT-injected CD11c.DOG (day 4) and littermate controls were incubated in vitro with purified mAb against NK1.1. Antibody-coated NK cells were stained with calcium dye Fluo-4 (Life Technologies) and purified Goat-Anti-Mouse IgG (H + L) Polyclonal Antibody (Jackson Immunoresearch) was added as indicated in the figure for measurement of real-time iCa2+ mobilization by flow cytometry. Receptor induced iCa2+ mobilization was measured by determining the intensity of iCa2+ peak and the integration of iCa2+ over time.
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