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Recombinant murine il 2

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Recombinant murine IL-2 is a laboratory reagent that provides the cytokine interleukin-2 from the mouse. Interleukin-2 is a signaling protein that plays a role in the immune system.

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Lab products found in correlation

Brefeldin a, by BioLegend (2 mentions) Easysep mouse cd4 cd25 regulatory t cell isolation kit, by STEMCELL (2 mentions) Immunocult cd3 cd28 t cell activator, by STEMCELL (2 mentions) Immunocult xf t cell expansion medium, by STEMCELL (2 mentions) Recombinant murine il 4, by BioLegend (2 mentions) Easysep human cd4 cd127lowcd25 regulatory t cell isolation kit, by STEMCELL (2 mentions) Human il 2, by STEMCELL (2 mentions) Pma ionomycin, by BioLegend (2 mentions) Prestoblue cell viability reagent, by Thermo Fisher Scientific (1 mentions) Cd4 cre mice, by Jackson ImmunoResearch (1 mentions) Anti cd11a m17 4, by BioXCell (1 mentions) Easysep mouse cd8 t cell isolation kit, by STEMCELL (1 mentions) Easysep mouse t cell isolation kit, by STEMCELL (1 mentions) Z2 coulter counter, by Beckman Coulter (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions) Ionomycin, by Merck Group (1 mentions) Flat bottomed 96 well plate, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) High glucose dmem, by Thermo Fisher Scientific (1 mentions) Glutamine, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Recombinant IL-2 (18 citations) Murine IL-2 (3 citations)

8 protocols using recombinant murine il 2

1

Crbn Knockout Regulatory T Cell Culture

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FLP293T derived stable cell lines were cultured in DMEM supplemented with 10% dialyzed fetal bovine serum (FBS). Wildtype and Crbn−/− Jurkat cells were cultured in RPMI supplemented with 10% FBS and 1% penicillin/streptomycin in a 37°C incubator with 5% CO2. CD4+ CD25+ regulatory T cells were purified from spleens from wildtype or CrbnI391V/I391V mice using the EasySep Mouse CD4+ CD25+ Regulatory T cell Isolation Kit (StemCell Technologies). T cells were cultured in RPMI supplemented with 10% FBS, 1% penicillin/streptomycin, 1% non-essential amino acids, 1% sodium pyruvate, 2 mM GlutaMax, 10 mM HEPES, and 50 μM 2-mercaptoethanol, as well as 5 ng/ml recombinant murine IL-2 (Biolegend) and 20 ng/ml recombinant murine IL-4 (Biolegend). Human regulatory T cells were isolated from peripheral blood mononuclear cells using the EasySep Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (StemCell Technologies) and expanded for 12–14 days in the presence of 500 U/ml human IL-2, Immunocult CD3/CD28 T cell activator (StemCell Technologies), and compound in ImmunoCult-XF T Cell Expansion Medium (StemCell Technologies). Cells were stimulated with 1X PMA/ionomycin (Biolegend) for 1h followed by the addition of brefeldin A (Biolegend) for another 3h.
Wang E.S., Verano A.L., Nowak R.P., Yuan J.C., Donovan K.A., Eleuteri N.A., Yue H., Ngo K.H., Lizotte P.H., Gokhale P.C., Gray N.S, & Fischer E.S. (2021). Acute pharmacological degradation of Helios destabilizes regulatory T cells. Nature chemical biology, 17(6), 711-717.
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2

Quantifying IL-2 Bioactivity in CTLL-2 Cells

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CTLL-2 cells (American Type Culture Collection #TIB-214) were maintained in RPMI 1640 with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin with 16 ng/ml recombinant murine IL-2 (Biolegend). For assessing IL-2 bioactivity, cells were placed in flat-bottom 96 well plates at 1x105 cells/well in 100 μl of medium. Diluted samples and standards were added in 100 μl to the wells and the plates were incubated at 37 °C in a 5% C02 atmosphere for 24 hours. PrestoBlue Cell Viability Reagent (Thermo) was used to measure metabolic activity in the wells according to the manufacturer’s instructions. The equivalent amount of bioactive IL-2 in the unknown samples was back-calculated by comparing to the standard curve.
Koshy S.T., Zhang D.K., Grolman J.M., Stafford A.G, & Mooney D.J. (2017). Injectable nanocomposite cryogels for versatile protein drug delivery. Acta biomaterialia, 65, 36-43.
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3

Crbn Knockout Regulatory T Cell Culture

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FLP293T derived stable cell lines were cultured in DMEM supplemented with 10% dialyzed fetal bovine serum (FBS). Wildtype and Crbn−/− Jurkat cells were cultured in RPMI supplemented with 10% FBS and 1% penicillin/streptomycin in a 37°C incubator with 5% CO2. CD4+ CD25+ regulatory T cells were purified from spleens from wildtype or CrbnI391V/I391V mice using the EasySep Mouse CD4+ CD25+ Regulatory T cell Isolation Kit (StemCell Technologies). T cells were cultured in RPMI supplemented with 10% FBS, 1% penicillin/streptomycin, 1% non-essential amino acids, 1% sodium pyruvate, 2 mM GlutaMax, 10 mM HEPES, and 50 μM 2-mercaptoethanol, as well as 5 ng/ml recombinant murine IL-2 (Biolegend) and 20 ng/ml recombinant murine IL-4 (Biolegend). Human regulatory T cells were isolated from peripheral blood mononuclear cells using the EasySep Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (StemCell Technologies) and expanded for 12–14 days in the presence of 500 U/ml human IL-2, Immunocult CD3/CD28 T cell activator (StemCell Technologies), and compound in ImmunoCult-XF T Cell Expansion Medium (StemCell Technologies). Cells were stimulated with 1X PMA/ionomycin (Biolegend) for 1h followed by the addition of brefeldin A (Biolegend) for another 3h.
Wang E.S., Verano A.L., Nowak R.P., Yuan J.C., Donovan K.A., Eleuteri N.A., Yue H., Ngo K.H., Lizotte P.H., Gokhale P.C., Gray N.S, & Fischer E.S. (2021). Acute pharmacological degradation of Helios destabilizes regulatory T cells. Nature chemical biology, 17(6), 711-717.
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4

Modulation of Regulatory T Cells via IL-2/IL-2mAb and LFA-1 Signaling

Cited 2 times
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All animal work was approved by the Institutional Animal Care and Use Committee of the University of California, San Diego. All mice were housed in specific pathogen-free conditions prior to use. Cd4Cre mice were purchased from Jackson Laboratories, and Tln1fl/fl and Foxp3GFP mice have been described previously (6 (link), 32 (link)–34 (link)). For administration of IL-2/IL-2mAb complexes, 2μg of recombinant murine IL-2 (Biolegend) was combined with 10μg IL-2 monoclonal antibody JES6-1 (Bio X Cell), diluted to a volume of 200μL in PBS and incubated for 30 minutes at 37°C before intraperitoneal (i.p.) injection, as previously described (35 (link), 36 (link)). To block LFA-1 signaling, mice were treated with 100μg anti-CD11a (M17/4) (Bio X Cell), 100μg anti-CD18 (M18/2) (Bio X Cell) and 200μg anti-ICAM-1 (YN1/1.7.4) or isotype control in PBS via i.p. injection twice weekly for three weeks.
Klann J.E., Remedios K.A., Kim S.H., Metz P.J., Lopez J., Mack L.A., Zheng Y., Ginsberg M.H., Petrich B.G, & Chang J.T. (2017). Talin plays a critical role in the maintenance of the regulatory T cell pool. Journal of immunology (Baltimore, Md. : 1950), 198(12), 4639-4651.
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5

Isolation and Activation of Murine T Cells

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Peripheral T cells were isolated from the spleens of 6–12-week-old mice via negative selection using the EasySep Mouse T-cell Isolation Kit or the EasySep Mouse CD8+ T-cell Isolation Kit (STEMCELL) and activated with anti-CD3/CD28 beads (Miltenyi T-cell Activation/Expansion Kit, mouse) at a 1:1 bead:cell ratio in the presence or absence of 25 U/mL recombinant murine IL-2 (BioLegend), as noted in the figure legends. Activated T cells were maintained at a density of 1 × 106 cells/mL, and the size of the cells was measured using a Z2 Coulter Counter (Beckman).
Holling G.A., Chavel C.A., Sharda A.P., Lieberman M.M., James C.M., Lightman S.M., Tong J.H., Qiao G., Emmons T.R., Giridharan T., Hou S., Intlekofer A.M., Higashi R.M., Fan T.W., Lane A.N., Eng K.H., Segal B.H., Repasky E.A., Lee K.P, & Olejniczak S.H. (2024). CD8+ T cell metabolic flexibility elicited by CD28-ARS2 axis-driven alternative splicing of PKM supports antitumor immunity. Cellular and Molecular Immunology, 21(3), 260-274.
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6

Stimulation of Splenocytes for NK Cell Assays

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Flat-bottomed 96 well plates (Thermo Fisher Scientific) were coated with 5 μg of α-NKR-P1C (clone PK136, BD Biosciences) in 100μL PBS overnight. The following day plates were washed 3 × and 1 × 106 splenocytes per well were stimulated for 5 h in RPMI + 5% FBS in the presence of 1:1000 Golgi Stop (BD BD Biosciences), 1000 IU/mL of recombinant murine IL-2 (BioLegend), and α-CD107a-AF647 (0.025 μg/well). Some cells were stimulated as above with the addition of 400 nM Phorbol myristate acetate (PMA, Sigma-Aldrich) and 3.35 μM ionomycin (Sigma-Aldrich).
Galpin K.J., Rodriguez G.M., Maranda V., Cook D.P., Macdonald E., Murshed H., Zhao S., McCloskey C.W., Chruscinski A., Levy G.A., Ardolino M, & Vanderhyden B.C. (2024). FGL2 promotes tumour growth and attenuates infiltration of activated immune cells in melanoma and ovarian cancer models. Scientific Reports, 14, 787.
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7

Murine CD8+ T-cell Activation and Expansion

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Mouse splenic CD8+ T-lymphocytes were isolated with anti-mouse CD8α microbeads (Miltenyi) on a MACS column. CD8+ T-lymphocytes were activated with plate-bound αCD3 (5 μg/ml, Biolegend) and soluble αCD28 (1μg/ml, Biolegend) for 48 h. For activation of OT-I CD8+ T-cells, total splenocytes from OT-I mice were cultured with SIINFEKL peptide (Sigma). Culture media was RPMI-1640 containing 2 mM glutamine (Gibco), 10% FBS, 25 mM HEPES, 1% penicillin-streptomycin (Gibco), 50 μM β-mercaptoethanol (Gibco). Following activation, CD8+ T-lymphocytes were expanded for 5 days in the presence of 20 ng/ml recombinant murine IL-2 (Biolegend) and cultured under 21%, 5% or 1% oxygen conditions. For hypoxia experiments, cells were transferred into a Ruskinn Sci-tive hypoxia workstation.
LLC, MS-1 and B16-F10 cell lines were cultured at 37 C with high glucose DMEM (Gibco) supplemented with 10% FBS. B16-OVA culture media was supplemented with 0.8 mg/ml of G418 (Gibco).
Palazon A., Tyrakis P.A., Macias D., Veliça P., Rundqvist H., Fitzpatrick S., Vojnovic N., Phan A.T., Loman N., Hedenfalk I., Hatschek T., Lövrot J., Foukakis T., Goldrath A.W., Bergh J, & Johnson R.S. (2017). An HIF-1α/VEGF-A Axis in Cytotoxic T Cells Regulates Tumor Progression. Cancer Cell, 32(5), 669-683.e5.
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8

Tumor-Infiltrating Lymphocyte Activation Assay

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Minced fragments of fresh MC38, LLC, or MOC1 tumors were incubated in RPMI 1640 media supplemented with glutamine (2 nM), HEPES (25 mM), 1 mM sodium pyruvate, 10 μM β-mercaptoethanol, 5% FBS, and recombinant murine IL2 (100 U/mL; Biolegend) for 72 hours. Untouched TILs were enriched to >95% purity with negative magnetic sorting (Pan T Cell Isolation Kit II, AutoMACSpro, Miltenyi Biotec). Antigen-presenting cells (APCs; whole splenocytes from naïve, WT B6 mice from Taconic, irradiated with 50 Gy) were pulsed for one hour with peptides of interest: class I-restricted antigen p15E604–611 (H-2Kb-restricted KSPWFTTL) (20 ), survivin/Birc557–64 (H-2Kb-restricted QCFFCFKEL) (21 (link)), Twist125–133 (H-2Db-restricted TQSLNEAFA), and Trp53232–240 (H-2Db-restricted KYMCNSSCM) (22 (link)). Peptides were synthesized by Peptide2.0. Antigen-pulsed APCs and TILs were co-incubated for 24 hours at a 3:1 APC:TIL ratio. Supernatants (50 μL) were analyzed for IFNγ production by ELISA (Mouse IFNγ Quantikine ELISA kit, R&D) per manufacturer recommendations. TILs alone, APCs alone, and peptide stimulations with ovalbumin257–264 (H-2Kb-restricted SIINFEKL) and VSV-N52–59 (H-2Db-restricted RGYVYQGL) were used as controls.
Nagaya T., Friedman J., Maruoka Y., Ogata F., Okuyama S., Clavijo P.E., Choyke P.L., Allen C, & Kobayashi H. (2019). Host immunity following near-infrared photoimmunotherapy is enhanced with PD-1 checkpoint blockade to eradicate established antigenic tumors. Cancer immunology research, 7(3), 401-413.
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