Mil 23

Manufactured by R&D Systems

Sourced in United States

The MIL-23 is a laboratory instrument designed for the analysis and measurement of various parameters. It features precise data acquisition and processing capabilities, enabling accurate and reliable results. The core function of the MIL-23 is to provide researchers and scientists with a versatile tool for their analytical needs.

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Lab products found in correlation

8 protocols using mil 23

Comprehensive Flow Cytometry Panel for Mouse T Cells

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Flow cytometry monoclonal antibodies against mouse antigens (CD4, clone RM4-5; CD8a, clone 53–6.7; CD62L, clone MEL-14; IFN-γ, clone XMG1.2; IL-2, clone JES6-16E3; Ifnar1, clone MAR1-5A3; PD-1, clone RMP1-30; T-bet, clone 4B10) were obtained from Biolegend. Tim-3 monoclonal antibody (clone 5D12) was a gift from V. Kuchroo. Carboxyfluorescein succinimidyl ester (CFSE) was obtained from Biolegend. The following recombinant cytokines and antibodies were used for in vitro T cell cultures: mIFN-β (PBL InterferonSource, indicated concentrations), mIL-12 (R&D Biosystems, 10 ng mL^-1), mIL-2 (Miltenyi, 5 ng mL^-1), hTGF-β (Miltenyi, 3 ng mL^-1), mIL-6 (Miltenyi, 20 ng mL^-1), mIL-23 (R&D Biosystems, 20 ng mL^-1), anti-mIL-4 (BioXCell; clone 11B11, 10 μg mL^-1), anti-CD3 (eBioscience; Functional Grade Purified, clone 145-2C11, 2 μg mL^-1), anti-CD28 (Biolegend; LEAF-purified, clone 37.51, 2 μg mL^-1). Western blotting primary antibodies were obtained from BD Transduction Laboratories (Stat1, clone 42/Stat1, dilution 1:1000; Stat1 pY701, clone 14/P-STAT1, dilution 1:1000; Stat4 pY693, clone 42/Stat1, dilution 1:500), Cell Signaling (Stat4, clone C46B10, dilution 1:500) or Millipore (GAPDH, mAb374, dilution 1:1000). Anti-mouse or-rabbit secondary antibodies were obtained from Jackson Immunoresearch and were used at 1:10000.

Boivin N., Baillargeon J., Doss P.M., Roy A.P, & Rangachari M. (2015). Interferon-β Suppresses Murine Th1 Cell Function in the Absence of Antigen-Presenting Cells. PLoS ONE, 10(4), e0124802.

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Passive EAE Induction and Evaluation

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Active EAE was induced by subcutaneous injection of mice with 100 μg MOG_35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) emulsified in complete Freund’s adjuvant supplemented with 250 μg Mycobacterium tuberculosis extract H37Ra (Difco). Mice received 180 ng pertussis toxin (List Biological Laboratories) intraperitoneally on days 0 and 2. For passive EAE experiments, 2D2 CD4⁺ T cells were activated and transduced under the Th17-polarizing condition with either a control retroviral vector or retrovirus overexpressing the Runx dominant negative mutant. On day 2, mIL-23 (10 ng/ml; R&D System) was added to retrovirally transduced 2D2 CD4⁺ T cells and cells were expanded for additional 3 days. Five days after initial stimulation, cells were sorted for GFP expression. Retrovirally transduced 2D2 T cells (>99% GFP⁺) were reactivated on anti-CD3/CD28 coated plates (each at 2 μg/ml) for 48 hours and injected into C57BL/6 or Rag2−/−Il2rg−/− recipients (5×10⁶/mouse intravenously). Mice were monitored daily for the development of EAE according to the following criteria: 0, no disease; 1 limp tail; 2, hind limb weakness/partial paralysis; 3, complete hind limb paralysis; 4, front and hind limb paralysis; 5, moribund state.

Wang Y., Godec J., Ben-Aissa K., Cui K., Zhao K., Pucsek A.B., Lee Y.K., Weaver C.T., Yagi R, & Lazarevic V. (2014). The transcription factors T-bet and Runx are required for the ontogeny of pathogenic interferon-γ-producing T helper 17 cells. Immunity, 40(3), 355-366.

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Isolation and Differentiation of Naive CD4+ T Cells

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Pooled cells from spleen and lymph nodes were first enriched for CD4⁺ T cells with Magnisort mouse CD4 T cell enrichment kit (eBioscience 8804-6821-74) and then stained and sorted for CD4⁺ CD25⁻ CD44^lowCD62L^high naive T cells. Sorted naive CD4⁺ T cells were used for in vitro cultures in IMDM (Life Science 12440-053) supplemented with β-mercaptoethanol, 10% fetal bovine serum and 1% penicillin-streptomycin. For survival experiment, naive T cells were cultured in the presence or absence of mIL-7 (20 ng/ml). For T cell activation, naive T cells were plated on cell culture plates pre-coated with goat anti-hamster IgG (MP Biomedicals, 0856984) and in the presence of hamster anti-mouse CD3ε (eBioscience 16-0031-86) and hamster anti-mouse CD28 (eBioscience 16-00281-86) antibodies and differentiated into different subsets using reagents and recipes listed in the table below: mIL-2 (Biolegend 575404), mIL-12 (Biolegend, 577004), mIL-4 (Biolegend 574304), mTGFβ1 (Biolegend 736102), mIL-6 (Biolegend 575702), αIFNγ (Biolegend 505812), αmIL-4 (Biolegend 504108), αmIL-6 (Biolegend 501110), mIL-1 (Peprotech 211-11B), mIL-23 (R&D systems 1887 CF), mIL-7 (Biolegend 577806). For the culture of pathogenic Th17 cells see EAE induction by adoptively transfer of Th17 cells.
Final Concentrations of Differentiation Antibodies and Cytokines.

Swan G., Geng J., Park E., Ding Q., Zhou J., Walcott C., Zhang J.J., Huang H.I., Hammer G.E, & Wang D. (2021). A Requirement of Protein Geranylgeranylation for Chemokine Receptor Signaling and Th17 Cell Function in an Animal Model of Multiple Sclerosis. Frontiers in Immunology, 12, 641188.

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Murine Th17 Cell Differentiation

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C57BL/6 mice (Yangzhou University Comparative Medical Research Center, Yangzhou, China) spleens were teased through sterilized 70 μm cell strainers (BD Biosciences, San Jose, CA, USA) to obtain single-cell suspensions in IMDM medium (Gibco, Grand Island, NY, USA) containing 10% FBS (Gibco, Grand Island, NY, USA). RBC lysis buffer (BD Pharmingen, San Diego, CA, USA) lysed red blood cells. Anti-CD4 magnetic beads (Miltenyi biotech, Bergisch Gladbach, Germany) was used to purify CD4⁺ T cells. Mixed lymphocyte were stained with APC-adjusted anti-mouse CD4 antibody (BD Biosciences, San Jose, CA, USA) to detect the purity of CD4⁺ T cells. For mouse Th17 cell differentiation, naïve CD4⁺T cells were seeded in 24 well plates at 10⁶/well and stimulated with plate-bound 1 μg/mL anti-mCD3 and 1 μg/mL soluble anti-mCD28 (eBioscience, San Diego, CA, USA) under Th17 polarizing conditions: 1 ng/mL mTGF-β, 10 ng/mL mIL-6, and 10 ng/mL mIL-23 (R&D, Minneapolis, MN, USA) for 72 h. Simultaneous treatment with different concentrations of rhIL-23R-CHR/Fc protein. The endotoxin was removed for all proteins with Endotoxin affinity Resin (Genscript, Piscataway, NJ, USA) before assays.

Gao Y., Bian Z., Xue W., Li Q., Zeng Y., Wang Y., Tang L., Tang T., Gao X, & Guo W. (2019). Human IL-23R Cytokine-Binding Homology Region-Fc Fusion Protein Ameliorates Psoriasis via the Decrease of Systemic Th17 and ILC3 Cell Responses. International Journal of Molecular Sciences, 20(17), 4170.

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Naïve 2D2 TCR-transgenic CD4+ T Cell Differentiation

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Naïve 2D2 TCR-transgenic CD4⁺ T cells from spleen and lymph nodes of 2D2 wild-type or 2D2 Tbx21^−/− mice were electronically sorted (CD4⁺V_β11⁺CD62L^hi), and activated under T_H17 polarizing conditions with the following: anti-CD3 (2.5 μg/ml) (145-2C11, BioXCell), anti-IL-4 (20 μg/ml) (11B11, BioXCell), anti-IFN-γ (20 μg/ml) (XMG1.2, BioXCell), mIL-6 (30 ng/ml) (Miltenyi Biotec), hTGF-β1 (3 ng/ml) (Miltenyi Biotec) in the presence of irradiated wild-type splenocytes at 5:1 ratio. After 60 h of activation, mIL-23 (10 ng/ml; R&D Systems) was added. On day 5 of culture, cells were reactivated on plates pre-coated with 2 μg/ml of anti-CD3 and anti-CD28 (PV1, BioXCell), for an additional 48 h, prior to adoptive transfer.

Kwong B., Rua R., Gao Y., Flickinger J J.r., Wang Y., Kruhlak M.J., Zhu J., Vivier E., McGavern D.B, & Lazarevic V. (2017). T-bet-dependent NKp46+ innate lymphoid cells regulate the onset of TH17-induced neuroinflammation. Nature immunology, 18(10), 1117-1127.

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T Cell Differentiation Protocols

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Naïve CD4 T cells were isolated from mouse spleens and lymph nodes by magnetic bead separation (Miltenyi Biotec) following the manufacturers’ protocols. Cells were cultured in glucose-free RPMI (Gibco) supplemented with 10% heat-inactivated FBS (Corning), 1% penicillin/streptomycin (Gibco), 50μM 2-Mercaptoethanol (Gibco), and either 10mM glucose (Sigma) or 10mM galactose (Sigma). Naïve CD4 T cells were activated with plate-bound anti-CD3e (5μg/ml) and soluble anti-CD28 (2μg/ml) in the presence of mIL-1β (10 ng/ml; R&D Systems), mIL-23 (10ng/ml; R&D Systems), mIL-6 (50ng/ml; R&D Systems), and h TGF-β (5ng/ml; Peprotech) for T_H17 cell polarization; mIL-12 (10ng/ml; R&D Systems) for T_H1 cell polarization, or TGF-β (10ng/ml; Peprotech) and mIL-2 (10ng/ml; R&D Systems) for T_reg polarization. All cells were cultured at 37°C and 5% CO₂. T_H17 differentiation was verified on day 3 or 4, and cytokine expression on day 5, at which point cells were used for subsequent assays.

Hong H.S., Mbah N.E., Shan M., Loesel K., Lin L., Sajjakulnukit P., Correa L.O., Andren A., Lin J., Hayashi A., Magnuson B., Chen J., Li Z., Xie Y., Zhang L., Goldstein D.R., Carty S.A., Lei Y.L., Opipari A.W., Argüello R.J., Kryczek I., Kamada N., Zou W., Franchi L, & Lyssiotis C.A. (2022). OXPHOS Promotes Apoptotic Resistance and Cellular Persistence in TH17 cells in the Periphery and Tumor Microenvironment. Science immunology, 7(77), eabm8182.

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Cardiac γδT Cell Activation Assay

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Cardiac γδT cells were enriched from total cardiac cells using anti-PE Microbeads UltraPure (Miltenyi Biotec) after staining with PE-conjugated anti-mouse γδTCR (BD). Isolated cardiac γδT cells were transferred to 96-well Maxisorp plate (Nunc), which was coated overnight with 100 μl of 4-μg/ml anti-CD3 antibody (145-2C11; TONBO Biosciences) and cultured in the presence of 10-ng/ml mIL-1β (Peprotech) and 10-ng/ml mIL-23 (RD systems). After 48 h, the supernatants were collected and assayed for IL-17A production using LEGEND MAX^TM ELISA Kit (BioLegend) according to the manufacturer’s instructions.

Nagasaka A., Mogi C., Ono H., Nishi T., Horii Y., Ohba Y., Sato K., Nakaya M., Okajima F, & Kurose H. (2017). The proton-sensing G protein-coupled receptor T-cell death-associated gene 8 (TDAG8) shows cardioprotective effects against myocardial infarction. Scientific Reports, 7, 7812.

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Naïve 2D2 TCR-transgenic CD4+ T Cell Differentiation

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