Cells (106/ml) were incubated in 96 well round-bottom plates in media alone (unstimulated) or stimulated in media containing PMA (50ng/ml, Sigma) and ionomycin (1μg/ml, Sigma) with monensin (2μM, Sigma) added at various time points during the 5.5–6 h stimulation at 37°C before analysis as previous described (12 (link)). In experiments where Th9 cells derived from WT and IL-2-deficient mice were co-cultured, PMA/ionomycin stimulation occurred in Eppindorf tubes under rotation at 37°C. Blocking antibodies to CD25 (3C7, 1μg/ml, Biolegend) were added 15 min prior to PMA/ionomycin stimulation and blocking antibodies to IL-2 (JES6-1A12 or S4B6-1, Biolegend or BioXcell), TNFα (MP6-XT22, Biolegend) and IL-4 were added (all antibodies used at 20 μg/ml) during stimulation as indicated. Real time PCR for analysis of gene expression, pSTAT protein staining, and retroviral transduction of primary cells was performed using standard methods as previously described (12 (link)).
Jes6 1a12
Manufactured by BioLegend
The JES6-1A12 is a mouse anti-mouse IL-6 antibody produced by BioLegend. It is a purified monoclonal antibody that can be used for the detection of mouse IL-6 in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (2 mentions)
Rabbit anti hamster igg, by MP Biomedicals (2 mentions)
Il 15, by Thermo Fisher Scientific (2 mentions)
Transit 293, by Mirus Bio (2 mentions)
Ionomycin, by BioLegend (1 mentions)
Monensin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by BioLegend (1 mentions)
Mp6 xt22, by BioLegend (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Recombinant mouse il 33, by BioLegend (1 mentions)
D0564, by Merck Group (1 mentions)
Recombinant mouse il 2, by Thermo Fisher Scientific (1 mentions)
Rat igg2a clone 2a3, by BioXCell (1 mentions)
Ril 33, by R&D Systems (1 mentions)
5 protocols using jes6 1a12
1
Th9 Cell Differentiation Assay
2
Regulatory T cell regulation in autoimmunity
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DT (D0564; Sigma-Aldrich) was injected i.p. into Foxp3DTR+ and Foxp3DTR− littermates at 30 ng/g of body weight. The regimens for DT administration are depicted in the corresponding figure. For the adoptive transfer of T reg cells into Foxp3DTR+ perinates, 0.4 × 106 TCRβ+CD4+Foxp3+ cells from lymph nodes of 20- to 25-d-old Aire+/+ or Aire−/− Foxp3-IRES-GFP mice were transferred into 8-d-old recipients. For the treatment of Aire+/+ mice with IL-2–αIL-2 mAb complexes, recombinant mouse IL-2 (PeproTech) was incubated with an αIL-2 mAb (JES6-1A12; BioLegend) at room temperature for 10 min before the injection. Mice were injected with 0.02 ml cytokine–antibody complexes containing 2.5 µg/ml IL-2 and 82.5 µg/ml αIL-2 mAb. Recombinant mouse IL-33 (BioLegend) was injected at 12.5 µg/kg; control mice were injected with an equal volume of PBS. αPD-1 mAb (29F.1A12) was injected at 3 mg/kg; control mice were injected with an equal amount of an isotype-matched control mAb (2A3; both from BioXCell). Measurement of blood glucose levels in mice treated with αPD-1 or IL-33 was performed on blood from the lateral tail vein.
3
Retrovirally Transducing Activated CD8+ T cells
4
Retrovirally Transducing Activated CD8+ T cells
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Naive CD8+ T cells were cultured in RPMI supplemented with 10% fetal bovine serum (Life Technologies) in the presence of soluble anti-CD3 (145-2C11, Biolegend) and anti-CD28 (37.51, Bio × Cell) at 0.1 μg/ml and 1 μg/ml, respectively, unless otherwise specified, in multi-well tissue culture plates coated with rabbit anti-hamster IgG (MP Biomedicals). For retroviral transduction of activated T cells, viral supernatants were prepared by transfecting PlatE cells46 (link) with TransIT 293 (Mirus Bio) and primed T cells were spinoculated following overnight stimulation as described previously24 (link). To retrovirally transduce CD8+ T cells without TCR stimulation, naive CD8+ T cells were cultured in the presence of IL-7 (10 ng/ml, Peprotech) and IL-15 (100 ng/ml, Peprotech) for 2 days prior to spinoculation. For IL-2 blockade, 2 μg/ml of anti-IL-2 (JES6-1A12, Biolegend) was added to the culture.
5
In vivo murine model of airway inflammation
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Mice were anesthetized by isoflurane inhalation followed by a single i.n. administration of 50 µl of A. alternata extract (12.5 µg; Greer Laboratories) or recombinant IL-33 (rIL-33, 1 µg; house-made [Cayrol et al., 2018 (link)]), and/or recombinant TL1A (rTL1A, 5 µg; R&D Systems) in PBS. Lungs and BAL fluids were collected at different time points (15 min, 1 h, 3 h, 6 h, 14 h, 24 h, 48 h) after the single i.n. treatment with A. alternata or cytokines for flow cytometry, Western blot, qPCR, and ELISA analyses. In some experiments, mice were treated by a single i.n. administration of A. alternata extract (12.5 µg), coadministrated with two doses (10 µg i.n. at t-12 h and 10 µg i.n. at t0) of the function-blocking anti-TL1A mAb L4G6 (Fang et al., 2008 (link)) (L4G6, # EMI006; Kerafast) or its isotype control (Armenian hamster IgG, clone PIP, #BE0260, RRID: AB_2687739; BioXCell) (Fig. 8, C–H ), or the function-blocking anti-IL-2 mAb JES6-1A12 (# 503706, RRID: AB_11150775; Biolegend) or its isotype control (rat IgG2a, clone 2A3, # BE0089, RRID: AB_1107769; BioXCell) (Fig. S5 G ). For in vivo imaging of endogenous IL-9high ILC2s (Fig. 5, K and L ), 100 µl of rIL-33 (1 µg) in PBS was injected i.p. for 6 consecutive days to expand lung ILC2s (Fig. S5 F ), before a single i.n. administration of rIL-33 (1 µg) and/or rTL1A (5 µg) in PBS.
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