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Anti il 2

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Anti-IL-2 is a laboratory product that detects the presence of interleukin-2 (IL-2), a cytokine involved in immune system regulation. It can be used for research purposes to measure IL-2 levels in samples.

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Lab products found in correlation

Anti tnf α, by BioLegend (8 mentions) Anti ifn γ, by BioLegend (6 mentions) Golgistop, by BD (6 mentions) Anti cd3, by BioLegend (5 mentions) Anti cd8α, by BioLegend (4 mentions) Anti il 4, by BioLegend (4 mentions) Anti cd4, by BioLegend (4 mentions) Anti il 10, by BioLegend (3 mentions) Permeabilizing buffer, by BD (3 mentions) Celltrace violet, by Thermo Fisher Scientific (3 mentions) Ionomycin, by Merck Group (2 mentions) Anti cd8, by BioLegend (2 mentions) Anti mouse anti ifnγ, by BioLegend (2 mentions) Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (2 mentions) Lsrfortessa flow cytometer, by BD (2 mentions) Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions) Phorbol myristate acetate (pma), by Thermo Fisher Scientific (1 mentions) Anti cd3 cd28 beads, by Thermo Fisher Scientific (1 mentions) Recombinant human il 2, by R&D Systems (1 mentions) Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)

17 protocols using anti il 2

1

T Cell Expansion and Cytokine Profiling

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Human CD45+/CD3+ sorted cells were cultured in RPMI 1640 (Gibco), Glutamax (Life Technologies) with 10% Foetal Bovine Serum (FBS, Life Technologies), 1% Penicillin/Streptomycin (Life Technologies) and supplemented with human recombinant IL2 (50 U/mL, R&D system) and IL-7 (5 ng/mL, Invitrogen) up to 28 days in 96, 48 and 24 wells (Falcon). For expansion, anti-CD3/CD28 beads (Thermofisher) were added in the proportion of 1:1.
Cytokine production was assessed by Phorbol Myristate Acetate (PMA) and Ionomycin (Io) stimulation assay: human CD45+/CD3+ sorted T cells and freshly isolated human thymocytes were incubated in RPMI, supplemented with 20% Human Serum Heat Inactivated (SIGMA-ALDRICH), Protein Transport Inhibitor Cocktail (500X, eBioscience), PMA (40 ng/mL, SIGMA-ALDRICH) and Io (4 μg/mL, SIGMA-ALDRICH) for 6 h at 37 °C. T cells were then washed with HBSS solution and stained for CD3, CD4 and CD8 (BioLegend), as well as APC-Cy7 fixable viability dye (Invitrogen), before fixation and permeabilisation with intracellular staining buffer kit (BioLegend) and intracellular staining with antibodies anti- IFNγ, anti-TNFα and anti-IL2 (BioLegend). Expanded and unstimulated T cells were used to set the FACS gates.
Campinoti S., Gjinovci A., Ragazzini R., Zanieri L., Ariza-McNaughton L., Catucci M., Boeing S., Park J.E., Hutchinson J.C., Muñoz-Ruiz M., Manti P.G., Vozza G., Villa C.E., Phylactopoulos D.E., Maurer C., Testa G., Stauss H.J., Teichmann S.A., Sebire N.J., Hayday A.C., Bonnet D, & Bonfanti P. (2020). Reconstitution of a functional human thymus by postnatal stromal progenitor cells and natural whole-organ scaffolds. Nature Communications, 11, 6372.
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2

Proliferation and Cytokine Analysis of Antigen-Specific CD4+ T Cells

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Cell proliferation was performed by labeling CD4+ T cells with 2uM CellTrace Violet (CTV, ThermoFisher) for 5 minutes at room temperature. After CTV labeling, cells were stained with tetramers, surface markers, enriched and sorted as above for 0407-YF50 tetramer+ cells and CD45RO+CD4+ T cells (400-500 cells/well). T cells were cultured with DMSO, PHA-M (1:100, ThermoFisher), or YF50 peptide (50ug/ml) together with 100,000 peripheral monocyte derived dendritic cells per well. CTV staining was analyzed after 5 days in culture. For cytokine analyses, tetramer enriched cells were collected in a 96-well plate and stimulated with phorbol myristate acetate (PMA, 5ng/mL, Sigma) and ionomycin (500ng/mL, Sigma) in the presence monensin (2uM, Sigma) and Brefeldin A (5ug/mL, Sigma) for 5-hours as previously described (Del Alcazar et al., 2019 (link); Wendel et al., 2018 ). After stimulation, cells were restained with tetramers, followed by intracellular cytokine staining with anti-TNF-a, anti-IL-2, and anti-IFN-γ antibodies (BioLegend) using BD Cytofix/Cytoperm Fixation/Permeabilization Kit according to manufacturer protocol (BD).
Pan Y.G., Aiamkitsumrit B., Bartolo L., Wang Y., Lavery C., Marc A., Holec P.V., Rappazzo C.G., Eilola T., Gimotty P.A., Hensley S.E., Antia R., Zarnitsyna V.I., Birnbaum M.E, & Su L.F. (2021). Vaccination reshapes the virus-specific T cell repertoire in unexposed adults. Immunity, 54(6), 1245-1256.e5.
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3

Comprehensive Immune Cell Profiling

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The following fluorophore-conjugated antibodies were used for flow cytometry: anti-CD8 (53–6.7), anti-Thy1.1 (OX-7), anti-Vα2 (B20.1), anti-CD45 (30-F11), anti-PD-1 (29F.1A12), anti-CXCR3 (CXCR3–173), anti-IFNγ (XMG1.2), anti-IL-2 (JES6–5H4), anti-TNFα (MP6-XT22), anti-CD28 (37.51), anti-Tbet (4B10) (Biolegend); anti-CD27 (LG.7F9), anti-LAG3 (C9B7W), anti-granzyme B (Ngzb), anti-Eomes (Dan11mag) (Thermo Fisher); anti-pCD3ζ (pY142) (K25–407.69), anti-CXCR5 (2G8) (BD Biosciences).
Snook J.P., Soedel A.J., Ekiz H.A., O’Connell R.M, & Williams M.A. (2020). Inhibition of SHP-1 expands the repertoire of antitumor T cells available to respond to immune checkpoint blockade.. Cancer immunology research, 8(4), 506-517.
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4

CD4+ and CD8+ T Cell Isolation and Stimulation

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T cells were purified from spleens of wild-type (WT) B6, B6 FIR, or BALB/c mice. Red blood cell-lysed, single-cell suspensions were incubated with anti-CD45R/B220, anti-CD16/CD32, anti–TER-119, anti–I-A/I-E, anti-CD11b, and anti-Ly6G/Ly6C obtained from BD Biosciences. For CD4+ T cells, anti-CD8α, or CD8+ T cells, anti-CD4, were included. Non-T cells were eliminated using Dynabeads (Life Technologies) following the manufacturer’s instructions. Control, LPS, or IL-33 DC were used as stimulators in 5 d MLR of CellTrace Violet (CTV; Life Technologies; Grand Island, NY)-labeled CD4+ T cells at a 1:10 DC:T cell ratio. Recombinant human (h) rhIL-2 (50 U/ml; Peprotech, Rocky Hill, NJ), IL-33 (10 ng/ml), or anti-IL-2 (10 μg/ml; JES6-1A12; BioLegend) were added to the MLR as indicated in the figures and figure legends.
Matta B.M., Lott J.M., Mathews L.R., Liu Q., Rosborough B.R., Blazar B.R, & Turnquist H.R. (2014). IL-33 is an unconventional alarmin that stimulates IL-2 secretion by dendritic cells to selectively expand IL-33R/ST2+ regulatory T cells. Journal of immunology (Baltimore, Md. : 1950), 193(8), 4010-4020.
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5

Cytokine Expression Profiling of Stimulated Splenocytes

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20 μg/mL PMA and 1 μg/mL of ionomycin were used to stimulate splenocytes in the presence of GolgiStop (BD Biosciences). The cells were then fixed and permeabilized using a Foxp3 kit (BD Biosciences). Intracellular cytokine staining was then performed using anti- IL-2 (BioLegend, clone JES6-5H4), anti-IL-10 (BioLegend, clone JES5-16E3), anti-IFNγ (BD, clone XMG1.2), anti-TNF (BioLegend, clone MP6-XT22), anti-IL-6 (Invitrogen, clone MP5-20F3), and anti-IL-1β (Invitrogen, clone NJTEN3).
Anyalebechi J.C., Sun Y., Davis C., Wagener M.E., Liang Z., Burd E.M., Coopersmith C.M, & Ford M.L. (2024). CD8+ T cells are necessary for improved sepsis survival induced by CD28 agonism in immunologically experienced mice. Frontiers in Immunology, 15, 1346097.
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6

Cytokine Profiling of Activated Lymphocytes

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Intracellular cytokine staining was performed as previously described (49 (link)). Surface mobilization of CD107a was measured as previously described (50 (link)). Briefly, isolated lymphocytes were cultured in RPMI (FCS 10%v/v) supplemented with anti-CD107a (Clone: Lamp-1, BioLegend), anti-CD107b (Clone: Mac-3, Biolegend), anti-CD28 (Clone 37.51; BioXCell); peptide, 2μM; GolgiPlug (BD, cat#: BDB555029) 1×10−3g/L; GolgiStop, cat#: 554724) 1×10−6M; for 6h, 37°C. The cells were then washed, stained with LIVE/DEAD fixable viability dye (ThermoFisher, cat#:), 30m, 20°C. Then stained with anti-CD3 (17A2, BioLegend) anti-CD8 (Clone 53–6.7, BioLegend), anti-CD4 (RM4–5, Biolegend), 30min, 4°C. The cells were washed, and suspended in Fix/Perm solution (BD, Cat#: 554715) then incubated for, 30min, 4°C. The cells were washed twice in Perm/Wash solution (BD, Cat#: 554715), suspended in Perm/Wash solution containing anti-IFNγ (Clone: XMG1.2, Biolegend), anti-IL-2 (Clone:JES6–5H4, Biolegend), anti-TNFα (Clone: MP6-XT22,Biolegend) then incubated, 30min, 20°C. Cells were then washed twice and suspended in PBS (PFA 1%w/v) and stored at 4°C until use.
Finnigan J.P., Newman J.H., Patskovsky Y., Patskovska L., Ishizuka A.S., Lynn G.M., Seder R.A., Krogsgaard M, & Bhardwaj N. (2023). Structural Basis for Self-Discrimination by Neoantigen-Specific TCRs. Research Square.
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7

Multiparametric Flow Cytometry Immunophenotyping

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PBMC were labeled with LIVE/DEAD fixable violet dye (L34955; Invitrogen), followed by surface antibody staining (CD4, Clone:RPA-T4; CD8, Clone:SK1; CD3, Clone:OKT3, CXCR5, clone:J252D4, Biolegend). After surface staining, cells were fixed and permeabilized using FOXP3/Transcription factor staining buffer set (#00-5523-00, Invitrogen) per manufacturer’s instructions. Fixed cells were stained for intracellular cytokines anti-IL-2 (Clone:MQ1-17412, Biolegend) and anti-interferon gamma (Clone:4S.B3, Biolegend). Data were collected by flow cytometric analysis on a LSR II (BD Biosciences) cytometer and analyzed using FlowJo (BD Bioscience).
Liu H., Aviszus K., Zelarney P., Liao S.Y., Gerber A.N., Make B., Wechsler M.E., Marrack P, & Reinhardt R.L. (2023). Vaccine-elicited B and T cell immunity to SARS-CoV-2 is impaired in chronic lung disease patients. medRxiv.
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8

Multiparametric Flow Cytometry of Immune Cells

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Flow cytometry on enriched Lin− cells was performed with a combination of the following fluorescence-conjugated mAbs (all from Miltenyi Biotec unless specified otherwise): APC-conjugated anti-NKp46 (29A1.4.9), anti-CD90.2 (30-H12), anti-Rorγ (t; REA278), anti-FcRIa (MAR-1), anti-CD4 (GK1.5); PE-conjugated anti-NK1.1 (PK136), anti-CD25 (7D4), anti-T1-ST2 (DIH9, from Biolegend), anti-CD117 (3C11), anti-IL-9 (RM9A4) and anti-IL-2 (JES6-5H4). For intracellular staining, phorbol 12-myristate 13-acetate (PMA)/ionomycin-stimulated cells were added of brefeldin, and then permeabilized with the CytoFix/CytoPerm kit (BD Biosciences) for intra-cytoplasmic detection of IL-9 and IL-2. Flow cytometry was done at 4 °C on cells first exposed to Fc receptor mAb (2.4G2). Cells were analysed with a BD LSRFortessa flow cytometer equipped with BD FACSDiva 7.0 software.
Moretti S., Renga G., Oikonomou V., Galosi C., Pariano M., Iannitti R.G., Borghi M., Puccetti M., De Zuani M., Pucillo C.E., Paolicelli G., Zelante T., Renauld J.C., Bereshchenko O., Sportoletti P., Lucidi V., Russo M.C., Colombo C., Fiscarelli E., Lass-Flörl C., Majo F., Ricciotti G., Ellemunter H., Ratclif L., Talesa V.N., Napolioni V, & Romani L. (2017). A mast cell-ILC2-Th9 pathway promotes lung inflammation in cystic fibrosis. Nature Communications, 8, 14017.
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9

Cytokine Profiling of Murine Splenocytes

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ICS assays were performed as previously described [46 (link)], with some modifications. Briefly, mouse splenocytes were added to the plate (2 × 106/well) and then stimulated with the peptide pool (2 μg/mL for individual peptide) for 5 h. PMA and ionomycin (Dakewe Bioengineering, China) were used as a positive control. The cells were incubated with GolgiStop (BD Biosciences, USA) for an additional 6 h at 37°C. Then, the cells were harvested and stained with anti-CD3 (BioLegend), anti-CD4 (BioLegend), and anti-CD8α (BioLegend) surface markers. The cells were subsequently fixed and permeabilized in permeabilizing buffer (BD Biosciences, USA) and stained with anti-mouse anti-IFNγ (BioLegend), anti-TNFα (BioLegend), anti-IL-2 (BioLegend), anti-IL-4 (BioLegend), and anti-IL-10 (BioLegend) antibodies. All fluorescent lymphocytes were gated on a FACSAria flow cytometer (BD Biosciences, USA).
Xu K., An Y., Li Q., Huang W., Han Y., Zheng T., Fang F., Liu H., Liu C., Gao P., Xu S., Liu X., Zhang R., Zhao X., Liu W.J., Bi Y., Wang Y., Zhou D., Wang Q., Hou W., Xia Q., Gao G.F, & Dai L. (2021). Recombinant chimpanzee adenovirus AdC7 expressing dimeric tandem-repeat spike protein RBD protects mice against COVID-19. Emerging Microbes & Infections, 10(1), 1574-1588.
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10

Activation of ILC2 Progenitors by IL-2

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Recombinant IL-2 (1 ug; BioLegend) and anti-IL-2 (10 ug; BioLegend) were preincubated at 37°C for 30 min to form IL-2 complexes (IL-2C). Mice were injected intraperitoneally with IL-2C, daily for 2 consecutive days. Seven days after the first IL-2C injection, the frequencies of ILC2 progenitors in the BM and ILC2s in the kidney were measured by FACS analysis. In the second set of experiments, IL-2C treated mice were injected intravenously with a BV650-labeled anti-CD45 antibody (BioLegend) 5 min before sacrifice on Day 7.
Ryu S, & Kim H.Y. (2023). Bone Marrow Progenitors and IL-2 Signaling Contribute to the Strain Differences of Kidney Innate Lymphoid Cells. Immune Network, 23(2), e15.
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