Il 23, by BioLegend - Product details

1

Stimulating Skin Cells with Cytokines

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Mouse dorsal skin cells were stimulated in culture with 40 ng/ml IL-23 (Biolegend, San Diego, CA, USA) alone or in combination with 40 ng/ml of IL-1β (eBioscience, Waltham, MA, USA) overnight. Supernatants were retained for cytokine analysis and cells were cultured with 10 mM Brefeldin A (Sigma Aldrich, Gillingham, UK) for 4-5 hours before staining for flow cytometric analysis. Alternatively, cells were stimulated with 50 ng/ml PMA and 500 ng/ml ionomycin (Sigma Aldrich, Gillingham, UK) and 10 mM Brefeldin A for 4 hours before staining for flow cytometric analysis. Similarly, small intestinal lamina propria cells were stimulated with 20 ng/ml IL- 23, IL-6, IL-2 (Biolegend, San Diego, CA, USA), and IL-1β in the presence of 10 μM Brefeldin A for 2 hours before PMA (50 ng/ml) and ionomycin (500 ng/ml) was added for a further 3 hours.
For co-culture of WT and CD200R1KO cells, dorsal skin cells were isolated and either the WT or CD200R1KO cells were labeled with 10 μM eF450-conjugated cell proliferation dye (eBioscience, Waltham, MA, USA) for 10 minutes in the dark at 37°C, then were washed and co-cultured with the unlabeled cells.

Linley H., Ogden A., Jaigirdar S., Buckingham L., Cox J., Priestley M, & Saunders A. (2023). CD200R1 promotes interleukin-17 production by group 3 innate lymphoid cells by enhancing signal transducer and activator of transcription 3 activation. Mucosal Immunology, 16(2), 167-179.

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CD4+ T Cell Differentiation Assay

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Lymphocytes were isolated from peripheral lymph nodes and spleens of age- and sex- matched mice and purified with CD4 microbeads (L3T4, Miltenyi Biotec). Purified CD4⁺ T cells were cultured in RPMI 1640 medium containing 10% FBS, 1% penicillin-streptomycin and 2.6 μl of β-mercaptoethanol and activated with plate-coated 2.5 μg/ml anti-CD3 (145–2c11, BioXCell) and 1 μg/ml anti-CD28 (37.51, BioXCell) antibodies. For Treg differentiation, designated doses of TGF-β (2 ng/ml) and IL-2 (40 U/ml) were added into the culture medium. Rapamycin and mTOR inhibitors (pp242) were used as indicated. For Th1 differentiation, 20 ng/mL IL-12 (Biolegend) and 20 μg/mL anti-IL-4 (11B11, BioXcell) were added to the culture. For pathogenic Th17 cell differentiation, 20 ng/mL IL-1β (Biolegend), 20 ng/mL IL-6 (Biolegend), 50 ng/mL IL-23 (Biolegend) and 10 μg/mL anti-IFNγ (XMG1.2, BioXcell) were added to the culture. For classical Th17 cell differentiation, 1 ng/mL TGF-β (Biolegend), 40 ng/mL IL-6 (Biolegend) and 10 μg/mL anti-IFNγ (XMG1.2, BioXcell) were added to the culture. For CFSE proliferation assay, a final concentration of 2 μM of carboxyfluorescein succinimidyl ester (CFSE) (C1157, Life Technologies) was used to label CD4⁺ T cells.

Chou W.C., Guo Z., Guo H., Chen L., Zhang G., Liang K., Xie L., Tan X., Gibson S.A., Rampanelli E., Wang Y., Montgomery S.A., Brickey W.J., Deng M., Freeman L., Zhang S., Su M.A., Chen X., Wan Y.Y, & Ting J.P. (2021). AIM2 in regulatory T cells restrains autoimmune diseases. Nature, 591(7849), 300-305.

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3

Naïve CD4+ T cell differentiation

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CD4⁺ T cells were enriched from PBMCs by negative selection using magnetic beads (Miltenyi) and naïve CD45RA⁺CCR7⁺CD4⁺ T cells were then isolated by cell sorting using a BD FACSAria™ cytometer. Naïve CD4⁺ T cells were seeded at a concentration of 5 × 10⁵ cells per well in a 96 well plate in complete medium and stimulated with anti-CD2/CD3/CD28 beads (Miltenyi) alone (T_H0 condition) or in the presence of human recombinant cytokines: For T_H1 conditions IL-12 (20ng/mL) (Biolegend); for T_H17 conditions IL-6 (20ng/mL), IL-23 (10ng/mL) (Biolegend) and TGF-β1 (5 ng/mL) (R&D Systems); for T_FH conditions IL-12 (2ng/mL), IL-23 (10 ng/mL) and TGF-β1 (5ng/mL)^{35 (link)}.

Weinacht K.G., Charbonnier L.M., Alroqi F., Plant A., Qiao Q., Wu H., Ma C., Torgerson T.R., Rosenzweig S.D., Fleisher T.A., Notarangelo L.D., Hanson I.C., Forbes L.R, & Chatila T.A. (2017). Ruxolitinib reverses Dysregulated T Helper Cell Responses and controls Autoimmunity caused by a Novel STAT1 Gain of Function Mutation. The Journal of allergy and clinical immunology, 139(5), 1629-1640.e2.

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Listeria Infection and T Cell Analysis

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Infection of mice with Listeria monocytogenes and subsequent analysis of T cell effector function were performed as previously described [27] (link), except that a cocktail of IL-23 (50 ng/ml; BioLegend), IL-1 (10 ng/ml; BioLegend), and Pam₃Cys (1 μg/ml; Invivogen) was used to elicit IL-17 production from both γδ-17 and DN-17 cells.

Samuelson E.M., Laird R.M., Papillion A.M., Tatum A.H., Princiotta M.F, & Hayes S.M. (2014). Reduced B Lymphoid Kinase (Blk) Expression Enhances Proinflammatory Cytokine Production and Induces Nephrosis in C57BL/6-lpr/lpr Mice. PLoS ONE, 9(3), e92054.

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5

Cytokine-Stimulated Skin and Intestinal Cell Analysis

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Mouse dorsal skin cells were stimulated in culture with 40 ng/mL IL-23 (Biolegend) alone, or in combination with 40 ng/mL IL-1β (eBioscience) overnight. Supernatants were retained for cytokine analysis and cells were cultured with 10 µM Brefeldin A (Sigma) for 4-5 hr before staining for flow cytometric analysis. Alternatively, cells were stimulated with 50 ng/mL PMA and 500 ng/mL ionomycin (Sigma) and 10 mM Brefeldin A for 4 hr before staining for flow cytometric analysis. Similarly, Small intestinal lamina propria cells were stimulated with 20 ng/mL IL-23, IL-6, IL-2 and IL-1β in the presence of 10 μM Brefeldin A for 2 hours, before PMA (50 ng/mL) and ionomycin (500 ng/mL) was added for a further 3 hours.
For co-culture of WT and CD200R1KO cells, dorsal skin cells were isolated and either the WT or CD200R1KO cells were labelled with 10 μM eF450-conjugated cell proliferation dye (Fischer Scientific) for 10 minutes in the dark at 37°C then were washed and co-cultured with the unlabelled cells.

Linley H., Ogden A., Jaigirdar S., Buckingham L., Cox J., Priestley M, & Saunders A. (2023). CD200R1 promotes interleukin-17 production by group 3 innate lymphoid cells by enhancing signal transducer and activator of transcription 3 activation. Mucosal Immunology, 16(2), 167-179.

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6

Generation and Analysis of Th17 Cells

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Human PBMCs were donated by Li Xia, who provided her written informed consent. All the cells were used in vitro only. The collection of human PBMCs was approved by the ethics committee of the Fudan Affiliated Minhang Hospital (2019-Pijian-010-01 K). Whole human blood was obtained from healthy volunteers, and peripheral blood mononuclear cells (PBMCs) were extracted from the whole blood using Ficoll (Fisher Scientific, Waltham, USA) centrifugation. CD3⁺ T cells purified from PBMCs were activated using anti-CD3/28 beads at a 1:1 ratio and polarized into type 17 T cells with human IL-1β (20 ng/ml, BioLegend, San Diego, CA), IL-6 (20 ng/ml, BioLegend, San Diego, CA), and IL-23 (50 ng/ml, BioLegend, San Diego, CA). After five days, the cytokine levels in the supernatant were determined using ELISA (Multisciences, Hangzhou, China). The cells were collected for flow cytometry analysis.

Xia L., Tian E., Yu M., Liu C., Shen L., Huang Y., Wu Z., Tian J., Yu K., Wang Y., Xie Q, & Zhu D. (2022). RORγt agonist enhances anti-PD-1 therapy by promoting monocyte-derived dendritic cells through CXCL10 in cancers. Journal of Experimental & Clinical Cancer Research : CR, 41, 155.

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7

Cytokine Profiling in Serum and Tissue

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Blood was obtained from the retro-orbital sinus, and serum was collected after blood coagulation. Wound edge tissue was collected in RIPA buffer and homogenized in the FastPrep instrument (Fastprep 24, Lysing Matrix D, MP Bio) per manufacturer’s protocol. The Bradford assay was performed to measure protein concentration of each sample. Serum and tissue IL-17A, IL-22, and IL-23 ELISAs were performed per the manufacturer’s instructions (Biolegend). Recombinant IL-17A, IL-22, or IL-23 was used as a positive control (Biolegend).

Wei J.J., Kim H.S., Spencer C.A., Brennan-Crispi D., Zheng Y., Johnson N.M., Rosenbach M., Miller C., Leung D.H., Cotsarelis G, & Leung T.H. (2020). Activation of TRPA1 Nociceptor Promotes Systemic Adult Mammalian Skin Regeneration. Science immunology, 5(50), eaba5683.

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8

Cytokine-Induced Keratinocyte Response

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Medium was removed, and new medium containing cytokines was used to stimulate keratinocytes for a variety of time points. Unless stated otherwise, IL-6, IL-23, IL-27, IL-35 (all from BioLegend), and IL-12 (Tonbo) were added to 1 to 2 ml of medium at a concentration of 100 ng/ml; IFN-α (BioLegend) or IFN-γ (PeproTech) was added to 1 to 2 ml of media at a concentration of 50 U/ml. For the cytotoxicity study, cells were suspended in CellTiter 96 AQueous One Solution Reagent and then incubated at 37°C for 1 to 4 hours in a humidified, 5% CO₂ atmosphere. Absorbance was read at 490 nm using a 24-well plate reader.

Kwock J.T., Handfield C., Suwanpradid J., Hoang P., McFadden M.J., Labagnara K.F., Floyd L., Shannon J., Uppala R., Sarkar M.K., Gudjonsson J.E., Corcoran D.L., Lazear H.M., Sempowski G., Horner S.M, & MacLeod A.S. (2020). IL-27 signaling activates skin cells to induce innate antiviral proteins and protects against Zika virus infection. Science Advances, 6(14), eaay3245.

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9

Quantifying Inflammatory Cytokine Levels

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Using blood samples collected from patients and control subjects as described in the flow cytometry section, we measured protein concentration with the bicinchoninic acid assay kit (BCA; Pierce, Appleton, WI, USA). The total protein concentration was adjusted to 1 mg/mL. The concentrations of TNF-α, CRP, IL-4, IL-6, IL-10, IL-17 (Sigma-Aldrich, St. Louis, MO, USA), IFN-γ (eBioscience, San Diego, CA), TGF-β (active; R&D Systems, Minneapolis, MN), and IL-23 (BioLegend, San Diego, CA, USA) were quantified by ELISA kits. Levels of TNF-α, CRP, IL-4, IL-6, IL-10, and IL-17 were measured within the range of the standard curve. The measurement range for the IFN-γ kit was 0.016 to 10 pg/mL, that for the TGF-β kit was 31.2 to 2000 pg/mL, and that for the IL-23 kit was 31.3 to 2000 pg/mL.

Jiang C., Kong W., Wang Y., Ziai W., Yang Q., Zuo F., Li F., Wang Y., Xu H., Li Q., Yang J., Lu H., Zhang J, & Wang J. (2016). Changes in the cellular immune system and circulating inflammatory markers of stroke patients. Oncotarget, 8(2), 3553-3567.

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10

CD4+ T Cell Differentiation Assay

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Lymphocytes were isolated from peripheral lymph nodes and spleens of age- and sex- matched mice and purified with CD4 microbeads (L3T4, Miltenyi Biotec). Purified CD4⁺ T cells were cultured in RPMI 1640 medium containing 10% FBS, 1% penicillin-streptomycin and 2.6 μl of β-mercaptoethanol and activated with plate-coated 2.5 μg/ml anti-CD3 (145–2c11, BioXCell) and 1 μg/ml anti-CD28 (37.51, BioXCell) antibodies. For Treg differentiation, designated doses of TGF-β (2 ng/ml) and IL-2 (40 U/ml) were added into the culture medium. Rapamycin and mTOR inhibitors (pp242) were used as indicated. For Th1 differentiation, 20 ng/mL IL-12 (Biolegend) and 20 μg/mL anti-IL-4 (11B11, BioXcell) were added to the culture. For pathogenic Th17 cell differentiation, 20 ng/mL IL-1β (Biolegend), 20 ng/mL IL-6 (Biolegend), 50 ng/mL IL-23 (Biolegend) and 10 μg/mL anti-IFNγ (XMG1.2, BioXcell) were added to the culture. For classical Th17 cell differentiation, 1 ng/mL TGF-β (Biolegend), 40 ng/mL IL-6 (Biolegend) and 10 μg/mL anti-IFNγ (XMG1.2, BioXcell) were added to the culture. For CFSE proliferation assay, a final concentration of 2 μM of carboxyfluorescein succinimidyl ester (CFSE) (C1157, Life Technologies) was used to label CD4⁺ T cells.

Chou W.C., Guo Z., Guo H., Chen L., Zhang G., Liang K., Xie L., Tan X., Gibson S.A., Rampanelli E., Wang Y., Montgomery S.A., Brickey W.J., Deng M., Freeman L., Zhang S., Su M.A., Chen X., Wan Y.Y, & Ting J.P. (2021). AIM2 in regulatory T cells restrains autoimmune diseases. Nature, 591(7849), 300-305.

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Il 23

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65 protocols using il 23

Stimulating Skin Cells with Cytokines

CD4+ T Cell Differentiation Assay

Naïve CD4+ T cell differentiation

Listeria Infection and T Cell Analysis

Cytokine-Stimulated Skin and Intestinal Cell Analysis

Generation and Analysis of Th17 Cells

Cytokine Profiling in Serum and Tissue

Cytokine-Induced Keratinocyte Response

Quantifying Inflammatory Cytokine Levels

CD4+ T Cell Differentiation Assay

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