CD4+ T cells were stained with Fixable Viability Dye (eBioscience, Nieuwegein, The Netherlands), and antibodies for PlexinB2 APC and its respective isotype control (both from R&D systems, Abingdon, United Kingdom). Alternatively, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, Nieuwegein, The Netherlands), and stained for IL-17A FITC, IL-22 APC, IFNγ PerCP-Cy5.5 (all from eBioscience, Nieuwegein, The Netherlands), IL-4 BV711, IL-13 PE and TNF BV421 (all from BD Biosciences, Vianen, The Netherlands). For proliferation analysis, CD4+ T cells were labeled with CellTrace Violet (1.5 µM, Thermo Fisher Scientific, Nieuwegein, The Netherlands) prior to culture. Samples were acquired on a BD LSR Fortessa (BD Biosciences), or on a BD FACSCanto (BD Biosciences, Vianen, The Netherlands) using the BD FACSDiva software (BD Biosciences, Vianen, The Netherlands). FlowJo software (BD Biosciences, Vianen, The Netherlands) was used for data analyses. All flow cytometry data is presented as the percentage of positive cells.
Il 13 pe
Manufactured by BD
Sourced in United States, France, Canada
The IL-13 PE is a fluorescently-labeled antibody that specifically binds to the interleukin-13 (IL-13) protein. It can be used in various immunoassays and flow cytometry applications to detect and quantify IL-13 expression.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by Merck Group (3 mentions)
Facscalibur, by BD (2 mentions)
Ifn γ apc, by BD (2 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (2 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Facscanto, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Fixable viability dye, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Il 22 apc, by Thermo Fisher Scientific (1 mentions)
Il 17a fitc, by Thermo Fisher Scientific (1 mentions)
Ifnγ percpcy5, by Thermo Fisher Scientific (1 mentions)
Il 4 bv711, by BD (1 mentions)
Tnf bv421, by BD (1 mentions)
Golgistop, by BD (1 mentions)
Cytofix cytoperm plus, by BD (1 mentions)
Cd3 apc cy7, by BD (1 mentions)
Cd4 pe cy5, by Thermo Fisher Scientific (1 mentions)
Cd69 ecd, by Beckman Coulter (1 mentions)
9 protocols using il 13 pe
1
Multiparametric Analysis of CD4+ T Cells
2
Splenic Cytokine Expression Profiling
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Splenic single cell suspensions were prepared and red cells were lysed with ACK lysing buffer (Gibco Life Technologies). Cells were initially blocked prior to staining with anti-CD16/32 antibody before surface staining for 20 min at 4°C with CD3-FITC (Clone 145-2C11), CD4-PerCP Cy 5.5 (Clone RM4-5). Intracellular cytokine staining was performed by ex-vivo re-stimulation as described previously [15] (link). Briefly, 5×106 splenocytes were plated with 1 µg/ml anti-CD28 (clone 37.51) and re-stimulated in a pre-coated well with anti-CD3 (10 µg/ml) (clone 145-2C11). Cells were incubated for 2.5 h followed by 2.5 h with GolgiStop (BD Biosciences). Cells were then surface stained, fixed and permeabilised with Cytofix/Cytoperm Plus for 20 min at 4°C before intracellular cytokine staining using IL-13 PE (Clone JES10-5A2) or IFNγ-APC (Clone XMG1.2) or isotype controls (all BD Biosciences). Cells were acquired using a FACSCalibur (BD Biosciences) and analysed using FlowJo Software.
3
Quantifying SARS-CoV-2 Spike-specific T cells
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Frequencies of S-specific memory T cells in blood and BAL were assessed using a re-stimulation assay as described previously using 2 μg/mL PepMix SARS-CoV-2 Spike overlapping peptides in DMSO (15mers with 11 amino acid overlap, JPT Peptide Technologies)51 (link). The antibody panel used for surface staining was: CD103 FITC (2G5, Beckman Coulter, cat# B49222, 1:50 dilution), CCR7 BV421 (G043H7, Biolegend, cat# 353208, 1:50 dilution), CD8a BV711 (RPA-T8, Biolegend, cat# 301044, 1:80 dilution), CD4 PE-Cy5.5 (S3.5, Invitrogen, cat# MHCD0418, 1:80 dilution) and CD45RA BV650 (5H9, BD, cat# 740608, 1:500 dilution), and the intracellular proteins were stained using: IL-21 AF647 (3A3-N2.1, BD, cat# 560493, 1:20 dilution), IL-13 PE (JES10-5A2, BD, cat# 559328, 1:33 dilution), IL-2 BV605 (MQ1-17H12, BD, cat# 564165, 1:50 dilution), IL-17A BV785 (BL168, Biolegend, cat# 512338, 1:67 dilution), CD69 ECD (TP.1.55.3, Beckman Coulter, cat# 6607110, 1:67 dilution), CD3 APC-Cy7 (SP34-2, BD, cat# 557757, 1:200 dilution) and IFNγ AF700 (B27, Biolegend, cat# 506516, 1:200 dilution). Acquisition was performed using BD LSRFortessa cell analyzer, and the data were analyzed using FlowJo software v.10.7.1 (FlowJo).
4
Multi-Cytokine Profiling of T Cells
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The cells were thawed and washed with PBS first and PBS/1% BSA later and then stained with surface antibodies for 30–60 minutes. Surface antibodies used were CD3 - Amcyan, CD4 - APC-H7 and CD8 - PE-Cy7 (all from BD Biosciences). The cells were washed and permeabilized with BD Perm/Wash™ buffer (BD Biosciences) and stained with intracellular cytokines for an additional 30 min before washing and acquisition. Cytokine antibodies used were IL-4 FITC, IL-5 APC and IL-13 PE (all from BD Pharmingen. Flow cytometry was performed on a FACS Canto II flow cytometer with FACSDiva software v.6 (Becton Dickinson). The lymphocyte gating was set by forward and side scatter and 100,000 gated lymphocyte events were acquired. Data were collected and analyzed using Flow Jo software. All data are depicted as Fo of CD4+ T cells expressing cytokine(s). Frequencies following media stimulation are depicted as baseline Fo while Fo following stimulation with antigens or PMA/ionomycin are depicted as net Fo (with baseline Fo subtracted).
5
Phenotyping of NK Cell Subsets
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The monoclonal antibodies CD3‐PerCP, CD56‐allophycocyanin(APC), CD158a‐FITC, CD158b‐PE, CD158e‐FITC, CD94‐FITC, CD62L‐FITC, CD54‐PE, CD11a‐FITC, CX3CR1‐FITC, CXCR4‐PE, CCR7‐PE, NKP30‐FITC, NKP46‐PE, IL13‐PE, IL10‐PE, TGF‐β‐PE and IFN‐γ‐FITC (BD Bioscience, Mountain View, CA, USA), and NKG2A‐PE (BeckmanCoulter, USA) and appropriate isotypes were used in individual 4‐colour flow cytometry assays to analyse the immunophenotype as well as the cytokine secretion of NK cells. Intracellular staining was performed using the Pharmingen Intracellular Staining Kit (BD Pharmingen, San Diego, CA, USA). The cells were incubated for 5 hours with phorbol myristate acetate (PMA) (40 ng/mL) plus ionomycin (2.5 μg/mL, all reagents from Sigma Chemical) to stimulate maximal IFN‐γ, IL‐13, TGF‐β and IL‐10 production; GolgiStop (0.7 μL/mL) was added to the sample during the last 4 hours to trap the protein in the cytoplasm. NK1, NK2, NK3 and NKr cells were identified as CD3−CD56+IFN‐γ+, CD3−CD56+IL‐13+,CD3−CD56+ TGF‐β+ and CD3−CD56+IL‐10+, respectively. The dose of NK1, NK2, NK3 and NKr cells was classified as the absolute number of NK1, NK2, NK3 and NKr cells infused in GBM and GPB (cells/kg). Data were analysed using a FACSCaliber 4‐colour flow cytometer (BD Biosciences) and FlowJo 7.6.1 software (Tree Star Inc, CA, USA).
6
Profiling Inflammatory Cytokines in Murine Lymph Nodes
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Cervical lymph nodes were sampled from 2-, 4-, and 6-month-old KCASP1Tg and WT mice. After washing, the cells were filtered through a mesh and incubated with ACK Lysing Buffer (Thermo Fisher Scientific, Waltham, MA, USA) to lyse the erythrocytes. Mononuclear cells were isolated and purified using density gradient centrifugation. They were then cultured in RPMI media supplemented with 50 ng/mL of phorbol myristate acetate (AdipoGen, San Diego, CA, USA) along with 1 µg/mL of ionomycin (Fujifilm, Tokyo, Japan) for 4 h. The LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) was used to exclude apoptotic and necrotic cells. The cultured mononuclear cells were stained with surface antibodies: CD4-Brilliant Violet 421, CD8-APC-Cy7, and γδ-TCR-APC (BD Biosciences, Franklin Lakes, NJ, USA) in cell surface staining buffer containing 0.1 M phosphate-buffered saline and 2% FCS (Biowest, Nuaillé, France) and then stained with IFNγ-Brilliant Violet 605 and TNFα-FITC, IL-4- Brilliant Violet 711, IL-13-PE, IL-17A-Brilliant Violet 786, and IL-17F-PerCp-Cy5.5 antibodies (BD Biosciences). The expression patterns of inflammatory cytokines were analyzed using a BD Lyric flow cytometer (BD Biosciences).
7
Multiparametric Flow Cytometry Assay
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For extracellular staining, harvested cells were washed and incubated in PBS containing 1% FBS containing the below fluorochrome-conjugated antibodies in a U-bottom 96-well plate. For intracellular cytokine staining, harvested cells were re-challenged in PMA and Ionomycin in the presence of Golgi-Plug (BD Biosciences). After 5 hours of incubation, the cells were fixed/permeabilized (eBioscience) and incubated with antibodies. The following antibodies were used for the flow cytometric assay: IFN-γ PE, IFN-γ APC, IL-13 PE, IL-17 APC (BD Bioscience), IL-2 APC (BD Pharmingen), Foxp3 PE (eBioscience). The gating strategy used has been described in Supplementary Fig. 4 . For cellular proliferation, Cell Trace CFSE cell proliferation kit (Invitrogen) was used per the manufacture’s manual.
8
Intracellular Cytokine Staining for Th2 Cells
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Intracellular staining to characterize primary Th2 cells was previously described in [42 , 43 (link)]. Briefly, cytokine staining for IL-4, IL-13 and IFNγ was performed following 4 h of stimulation with PMA (20 ng/mL) and ionomycin (1 μM) in the presence of Brefeldin A (10 μg/mL, all from Sigma Aldrich, Canada). Cells were fixed on ice (10 min) with paraformaldehyde (4%; Sigma Aldrich, Canada) and permeabilized on ice (10 min) with saponin (0.4%, Sigma Aldrich, Canada). Cells were incubated with at 4 °C (30 min) with IL-13-PE (Clone JES10-5A2, isotype rat IgG1κ PE), IFNγ-Alexa 647 (Clone B27, isotype mouse IgG1κ Alexa 647) or IL-4-Alexa 488 (Clone 8D4–8, isotype mouse IgG1κ Alexa 488) antibodies (BD Pharmingen, ON, Canada) and then washed with buffer. Fluorescence was assessed immediately using a FACS Calibur (Becton Dickson, ON, Canada) and data analyzed using FlowJo (Tree Star Inc., Ashland, OR, USA).
9
Treg Secretion Assay for Immune Profiling
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For the T-regulatory cell (Treg) secretion assay, peripheral blood mononuclear cells were thawed and rested 1 night in complete medium (RPMI-160, 10% fetal bovine serum, 1% glutamine, 1% penicillin/streptomycin; 5 million/ml). PBMCs were then cultured at 5 million/ml in complete medium and activated for 5 hours with phorbol 12-myritate 13-acetate (PMA, 50 ng/ml), ionomycin (1 µg/ml), and brefeldin A (1×) (Sigma Aldrich, St. Louis, MO) to block intracellular trafficking. The following antibodies and reagents were used for flow cytometry: CD3-BUV395 (UCHT1); CD25-BV786 (M-A251); CD4-BV650 (SK3); CD127-BV450 (HIL-7R-M21); IL-10-APC (JES3-19F1); IL-17A-PE (SCPL1362); IL-13-PE (JES10-5A2); IL-4-APC (MP4-25D2); and IfnG-FITC (B27) (BD Biosciences, Heidelberg, Germany); CD45RA-APCeFluor780 (HI-100); and FoxP3-PercpCy5.5
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