GMSCs were collected and suspended in cell staining buffer (0.5% BSA in PBS with 2 mM EDTA) followed by incubation with CD14 (PE), CD19 (PerCP-Cy5.5), CD29 (APC), CD34 (PE), CD44 (FITC), CD45 (PE), CD73 (PE), CD90 (FITC), and CD105 (PE) antibodies (Biolegend) in the dark at room temperature for 30 min. For intracellular staining, splenocytes from mice were first stained with CD3 (APC/Cy7), CD4 (FITC), and CD25 (APC) antibodies (Biolegend) and then fixed, permeabilized, and stained intracellularly for IL-4 (PE/Cy7) and IL-17 (PE). After staining, cells were washed twice with PBS and submitted to flow cytometric analysis (BD). Data were analyzed using the FlowJo 7.6 software.
Il 4 pe cy7
Manufactured by BioLegend
Sourced in United States
IL-4 (PE/Cy7) is a fluorochrome-conjugated antibody that detects the cytokine interleukin-4 (IL-4). It is a cell surface marker commonly used in flow cytometry applications to identify and quantify IL-4 expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd14 pe, by BioLegend (1 mentions)
Cd25 apc, by BioLegend (1 mentions)
Cd105 pe, by BioLegend (1 mentions)
Il 17, by BioLegend (1 mentions)
Flow cytometric analysis, by BD (1 mentions)
Cd19 percp cy5, by BioLegend (1 mentions)
Lsr fortessa cytometer, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Zombie aqua, by BioLegend (1 mentions)
Il 2 pe, by BioLegend (1 mentions)
Anti cd28, by BD (1 mentions)
Interleukin 2 (il 2), by Roche (1 mentions)
Anti cd8 percp cy5, by BioLegend (1 mentions)
Anti cd16 cd32, by BD (1 mentions)
Anti cd49d, by BD (1 mentions)
Ifn γ fitc, by BioLegend (1 mentions)
Cytofix cytoperm buffer, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Facsaria 3 sorter, by BD (1 mentions)
Cd11b pe, by Thermo Fisher Scientific (1 mentions)
4 protocols using il 4 pe cy7
1
Immunophenotyping of GMSCs and Splenocytes
2
Quantifying Influenza-Specific T-Cell Responses
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CD4+ and CD8+ T-cell recall responses were determined as described previously (36 (link), 37 (link)). The production of type 1 (IFN-γ, TNF-α, and interleukin 2 [IL-2]) and type 2 (IL-4) cytokines of T cells was detected. Splenocytes and cells from BAL fluid (n ≥ 3 fluid samples per group) were harvested at day 7 postinfection. After RBC lysis, isolated lymphocytes were stimulated by PR8 or HK68 virus and coincubated with anti-CD28, anti-CD49d (BD Biosciences), and IL-2 (Roche) for 6 h, followed by an incubation with GolgiPlug (containing brefeldin A; BD Biosciences) overnight. For splenocytes at 3 weeks after vaccination, PR8, HK68, H1N1/Brisbane/07 (H1N1), and HK/MPF461/07 (H5N2) viruses were used to stimulate the cells. Treated cells were first stained with Zombie Aqua (Biolegend) and then with T-cell markers (Fc block by anti-CD16/CD32 [BD Biosciences]; anti-CD4–allophycocyanin/Cy7 and anti-CD8–PerCP/Cy5.5 [Biolegend]). Stained cells were then fixed with Cytofix/Cytoperm buffer (BD), followed by intracellular cytokine staining (IFN-γ, FITC; TNF-α, allophycocyanin; IL-2, PE; and IL-4, PE/Cy7; Biolegend). Signal acquisition was performed on a BD LSR Fortessa cytometer, and data were analyzed with FlowJo.
3
Multicolor Flow Cytometry Protocol
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For surface and intracellular staining, the monoclonal antibodies listed were used: CD4 (PerCP) (RM4-5); CD192 (CCR2) (Alexa 647) (SA203G11) all from BioLegend (Biozol); CD8 (53-6.7); IL-4 (PE-Cy7) (11B11); IL-5 (PE) (TRFK5); IL-13 (Alexa 488) (eBio13A); IFN-γ (eFluor 450) (XMG1.2); CD154 (PE) (MR1); Foxp3 Alexa 488 (FJK-16s); GATA3 (eFluor 660) (TWAJ); Dead Cell Exclusion Marker (DCE) (efluor 780); DCE (efluor 506); Siglec F (PE) (E50-2440); T-bet (PE) (eBio4B10); IL-13 (eFluor 660) (eBio13A); CD11b (PE) (M1/70); F4/80 (PerCP-Cy5.5) (BM8); Ly-6G (Gr-1) (PE-Cy7) (RB6-8c5); Ly-6C (eFluor 450) (HK1.4); TNF-α (Alexa488) (MP6-XT22) all from eBioscience, San Diego, CA, USA.
For intracellular staining of cytokines and transcription factors cells were fixed and permeabilized using the fix/perm buffer kit (eBioscience, San Diego, CA, USA). FACSCantoII flow cytometer and FACSAriaIII sorter (both BD Bioscience, Heidelberg, Germany) were used for cell analysis. FlowJo software 10.2 was used for final analysis (Tree star Inc., Ashland, OR, USA).
For intracellular staining of cytokines and transcription factors cells were fixed and permeabilized using the fix/perm buffer kit (eBioscience, San Diego, CA, USA). FACSCantoII flow cytometer and FACSAriaIII sorter (both BD Bioscience, Heidelberg, Germany) were used for cell analysis. FlowJo software 10.2 was used for final analysis (Tree star Inc., Ashland, OR, USA).
4
Multiparametric Immune Profiling of Splenocytes
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Splenocytes were incubated with either 20 ng mL−1 phorbol myristate acetate (Sigma‐Aldrich) and 1 ug mL−1 ionomycin (Cell Signaling Technology), S protein‐derived peptides (GenScript), N protein‐derived peptides (GenScript), or control (no stimulation) in 1X Brefeldin A and 1X Monensin (Biolegend) solutions for 6 h at 37 °C. Next, the cells were washed with PBS and incubated for 30 min with Fixable Viability Dye eFluor 780 (eBioscience) in PBS (1:1000 dilution) at 4 °C. Cells were then washed once with PBS and twice with PBA before being stained overnight with fluorochrome‐conjugated antibodies (CD3‐FITC (Clone 145‐2C11), CD4‐PerCP‐Cy5.5 (Clone GK1.5), CD8‐AF700 (Clone 53–6.7), CD44‐BV605 (Clone IM7), Biolegend) in PBA at 4 °C. Cells were subsequently washed 3 times with PBA, fixed in 4% PFA, and then permeabilized using a Cyto‐Fast Fix/Perm Buffer Set (Biolegend) according to the manufacturer's protocol. Intracellular staining was performed by incubating the cells with fluorochrome‐conjugated antibodies (IL‐13‐PE (Clone: W17010B), IL‐4‐PE‐Cy7 (Clone: 11B11), IL‐17‐APC (Clone: TC11‐18H10.1), IL‐5‐BV421 (Clone: TRFK5), IFNγ‐BV510 (Clone XMG1.2), Biolegend) in permeabilization buffer for 30 min at 4 °C. Lastly, cells were washed 3 times with permeabilization buffer, resuspended in PBA, and analyzed using the Attune NxT flow cytometer (Thermo Fisher Scientific).
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