ILC2s were cultured in the presence of rm-IL-2 (10 ng/mL) and rm-IL-7 (10 ng/mL). When indicated, ILC2s were incubated with purified mouse C1q (10 μg/mL, Complement Technology) or with 15 μM of the SHP-1 inhibitor, known as Tyrosine Phosphatase Inhibitor 1 (TPI-1, MedChemExpress)39 (link),43 . The BD Biosciences Cytofix/Cytoperm kit was used and followed by intracellular staining with PE anti-p65 (clone 532301, R&D Systems), BV421 anti-AKT (pS473) (clone M89-61, BD Biosciences), PE-Cy7 anti-BCL-2 (clone 10C4, eBioscience) and unconjugated anti-SHP-1 (clone SR41-02, ThermoFisher) followed by Alexa Fluor 647 goat anti-rabbit IgG secondary antibody (Jackson ImmunoResearh). Intranuclear staining was performed using the Foxp3 Transcription Factor Staining Kit (ThermoFisher) according to the manufacturer’s instructions and APC anti-mouse Ki67 (SolA15, ThermoFisher) was used to assess proliferation. The CellTrace Violet Cell Proliferation Kit (ThermoFisher) was also used in some experiments, according to manufacturer’s instructions. In parallel, the levels of IL-5, IL-6, and IL-13 were measured in supernatants using Legendplex multiplex kits (BioLegend) and data were analyzed via the LEGENDplex data analysis software v8.0. GM-CSF was quantified using ELISA kit from BioLegend.
Legendplex multiplex kits
Manufactured by BioLegend
LegendPlex multiplex kits are designed for the simultaneous quantification of multiple analytes in a single sample. The kits utilize fluorescent-encoded beads and a flow cytometry-based platform to enable the rapid and sensitive detection of a variety of biological molecules, including cytokines, chemokines, and signaling proteins.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Tpi 1, by MedChemExpress (1 mentions)
Cytofix cytoperm kit, by R&D Systems (1 mentions)
Pe cy7 anti bcl 2 clone 10c4, by Thermo Fisher Scientific (1 mentions)
Foxp3 transcription factor staining kit, by Thermo Fisher Scientific (1 mentions)
Celltrace violet cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
Elisa max deluxe set mouse il 17a kit, by BioLegend (1 mentions)
4 protocols using legendplex multiplex kits
1
ILC2 Signaling Profiling and Cytokine Analysis
2
Therapeutic Targeting of PD-1 in ILC2s
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aILC2s were cultured (5 × 104 per mL) for 24 h in the presence of rm-IL-2 (10 ng mL−1) and rm-IL-7 (10 ng mL−1). When indicated, ILC2s were incubated in PD-L2 Fc or isotype-coated wells (5 μg mL−1, R&D systems). The levels of IL-5, IL-9, and IL-13 were measured in supernatants using Legendplex multiplex kits (BioLegend) and data were analyzed via the LEGENDplex data analysis software v8.0. Human ILC2s were sorted and cultured for 48 h with rh-IL-2 (20 ng mL−1), rh-IL-7 (20 ng mL−1), rh-IL-33 (20 ng mL−1), and PD-1 agonist or control isotype (10 μg mL−1). Human IL-5 and IL-13 were quantified in supernatants by ELISA (ThermoFisher Scientific). The PD-1 agonist has been fully characterized57 . Briefly, the antibody binds to human PD-1 with an equilibrium dissociation constant (KD) of 5 × 10−8 M; an association constant (ka) of ~3 × 104 l Ms−1 and a dissociation constant (kd) of ~3 × 10−3 l s−1. To obtain antibody please contact Janssen Pharmaceuticals.
3
Protein Quantitation and Cytokine Profiling
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The BCA Protein Assay was used for colorimetric quantitation of total protein in the BAL (ThermoFisher). The levels of cytokines were measured in the BAL and culture supernatants using Legendplex multiplex kits (BioLegend).
4
Multiplex Cytokine Profiling in Murine Lung
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