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.
Pe cy7 anti bcl 2 clone 10c4
Manufactured by Thermo Fisher Scientific
PE-Cy7 anti-BCL-2 (clone 10C4) is a flow cytometry reagent that binds to the BCL-2 protein. It is conjugated with the fluorescent dye Phycoerythrin-Cyanine 7 (PE-Cy7).
Automatically generated - may contain errors
Lab products found in correlation
Tpi 1, by MedChemExpress (1 mentions)
Cytofix cytoperm kit, by R&D Systems (1 mentions)
Foxp3 transcription factor staining kit, by Thermo Fisher Scientific (1 mentions)
Legendplex multiplex kits, by BioLegend (1 mentions)
Celltrace violet cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti cd3 145 2c11, by BioLegend (1 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions)
2 protocols using pe cy7 anti bcl 2 clone 10c4
1
ILC2 Signaling Profiling and Cytokine Analysis
2
Multiparameter Flow Cytometry Analysis
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The following antibodies were used for the flow cytometry analysis: phycoerythrin (PE)- and Cy7-conjugated anti-CD8 (clone 53-6.7; eBioscience), allophycocyanin (APC)-conjugated anti-CD3 145-2C11; BioLegend), fluorescein isothiocyanate (FITC)-conjugated-CD25 (clone PC 61; eBioscience), APC-anti-CD44 (clone IM7; eBioscience), PE-anti-CD62L (clone M1L-14; Tonbo), PE-anti-CD122 (clone TM-b1; eBioscience), PE-anti-CD127 (clone A7R34; BioLegend), PerCP- and Cy5.5-conjugated-ScaI (clone D7; eBioscience), APC-anti-KLRG1 (clone 2F1; BioLegend), PE-anti-NK1.1 (clone PK136, eBioscience), FITC-anti-PD-1 (clone J43; eBioscience), PE-anti-B7H1 (clone 10F.9G2), PerCP-Cy5.5-anti-CD45 (clone 30-F11; BioLegend), FITC-anti-CD90.1 (clone HIS51; eBioscience), APC-anti-Flag (clone L5; BioLegend), PE-Cy7-anti-Bcl-2 (clone 10C4; eBioscience), PE-anti-TCF1 (clone 14456S; Cell Signaling Technology), FITC-anti-CD95 (clone SA367H8, BioLegend), PE-anti-IFN-γ (clone XMG1.2; eBioscience), and APC-anti-IL-2 (clone JES6-5H4; eBioscience). For the CFSE dilution assay, T cells were labeled with 5 μM CFSE before being cultured. Dead cells were discriminated with the Fixable Viability Dye eFluor™ 780 (Thermo Fisher Scientific). The stained cells were analyzed with a cytoflex instrument (Beckman coulter). The data analysis was performed using FlowJo X software (Tree Star).
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