Mog35 55

Manufactured by Thermo Fisher Scientific

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The MOG35–55 is a laboratory equipment product that serves a core function. It is a device designed for specialized applications in the scientific research and analysis domains.

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7 protocols using mog35 55

Intracellular Cytokine Staining of T Cells

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For intracellular cytokine staining (ICS) assays, T cells isolated from spleen of mice or from in vitro cultures were stimulated for 1.5 h with 100 mg/ml MOG(35-55) or PMA (50 ng/ml, Thermo Fisher Scientific, USA) and ionomycin (500 ng/ml, Thermo Fisher Scientific), before Brefeldin A (10 μg/ml, eBioscience, USA) was added to the culture for 3.5 h more. As previously described [32 (link)], for surface staining, cells were harvested, washed, and stained for 30 min on ice with mixtures of fluorescently conjugated mAbs or isotype-matched controls. For ICS, cells were stained for surface molecules, fixed 20 min in IC Fixation buffer (Thermo Fisher Scientific), and incubated for 1 h in permeabilization buffer (Thermo Fisher Scientific) with appropriate mAbs of mice. Antibodies used in this study are listed in Supplementary Table 1. Cell phenotype was analyzed by flow cytometry on a flow cytometer (BD LSR II) (BD Biosciences, USA) or Attune NxT (Thermo Fisher Scientific). Data were acquired as the fraction of labeled cells within a live-cell gate and analyzed using FlowJo software (Tree Star). All gates were set on the basis of isotype-matched control antibodies.

Fu Y., Wang P., Zhao J., Tan Y., Sheng J., He S., Du X., Huang Y., Yang Y., Li J., Cai Y., Liu Y, & Hu S. (2021). USP12 promotes CD4+ T cell responses through deubiquitinating and stabilizing BCL10. Cell Death and Differentiation, 28(10), 2857-2870.

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Intracellular Cytokine Staining of T Cells

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For intracellular cytokine staining assays, T cells isolated from spleen or nervous system of mice, or from in vitro cultures were stimulated for 1.5 h with 100 mg/mL MOG(35-55) or PMA (50 ng/mL, Thermo Fisher Scientific, USA) and ionomycin (500 ng/mL, Thermo Fisher Scientific), before Brefeldin A (10 μg/mL, eBioscience, USA) was added to the culture for 3.5 h more. As previously described 37 (link), for surface staining, cells were harvested, washed, and stained for 30 min on ice with mixtures of fluorescently conjugated mAbs or isotype-matched controls. For intracellular cytokine staining (ICS), cells were stained for surface molecules, fixed 20 min in IC Fixation buffer (Thermo Fisher Scientific), and incubated for 1 h in permeabilization buffer (Thermo Fisher Scientific) with appropriate mAbs of mice. Antibodies used in this study are listed in Table S1. Cell phenotype was analyzed by flow cytometry on a flow cytometer (BD LSR II) (BD Biosciences, USA) or Attune NxT (Thermo Fisher Scientific). Data were acquired as the fraction of labeled cells within a live-cell gate and analyzed using FlowJo software (Tree Star). All gates were set on the basis of isotype-matched control antibodies.

Wang P., Zhao J., Tan Y., Sheng J., He S., Chen Y., Nie D., You X., Luo J., Zhang Y, & Hu S. (2023). RNF157 attenuates CD4+ T cell-mediated autoimmune response by promoting HDAC1 ubiquitination and degradation. Theranostics, 13(11), 3509-3523.

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Passive EAE Induction via Adoptive Transfer

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WT female 8- to 12-wk-old donor mice were immunized as described for active EAE but did not receive PTX. At DPI10, spleen and dLNs (inguinal, axillary, and brachial) were collected and passed through a 70-µm filter (Falcon) to obtain a single-cell suspension. Cells were incubated with 1× RBC lysis buffer (BioLegend) for 5 min at 4°C. After centrifugation (500 ×g for 5 min at 4°C), cells were cultured in complete T cell media (5 × 10⁶ cells/ml), DMEM (Corning) supplemented with 50 µg/ml MOG_35–55, 25 ng/ml IL-12, and 10 ng/ml IFN-γ (Thermo Fisher Scientific). After 72 h, CD4⁺ T cells were collected using the Dynabeads Untouched Mouse CD4 Cells Kit (Thermo Fisher Scientific) and injected into recipient mice (10⁶ cells/mouse i.p.). PTX was injected (200 ng/mouse i.p.) at days 0, 2, and 3 after CD4⁺ cell transfer. Clinical scoring and disease severity were assessed as above.

Mimouna S., Rollins D.A., Shibu G., Tharmalingam B., Deochand D.K., Chen X., Oliver D., Chinenov Y, & Rogatsky I. (2020). Transcription cofactor GRIP1 differentially affects myeloid cell–driven neuroinflammation and response to IFN-β therapy. The Journal of Experimental Medicine, 218(1), e20192386.

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Adoptive Transfer Model of EAE

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Donor C57BL/6 mice were actively immunized with 100 μg MOG_35–55 peptide emulsified in CFA (Difco). At day 9 after immunization, lymphocytes from the spleen, axillary, inguinal, and cervical lymph nodes were collected and cultured in vitro for 72–84 hr in complete T cell media (RPMI1640, 10% fetal bovine serum (GIBCO), 100 U/ml penicillin, 100 μg/ml streptomycin, 1X Glutamax (GIBCO), 1X MEM non-essential amino acid (GIBCO); 1mM sodium pyruvate (Sigma), 10mM HEPES (Lonza), and 50 μM β-mercaptoethanol). For T cell skewing, MOG_35–55 (20 μg/ml), rmIL-6 (20 ng/ml, Peprotech), rmIL-12p70 (3 ng/ml, Peprotech), rmIL-23 (20 ng/ml, R&D), and human TGFb (4 ng/ml, Peprotech) were added to the media. 40 million cells from cultured lymphocytes were transferred intraperitoneally to C57BL/6 or BAFF-Tg recipient mice. At day 2 post-transfer, recipients also received 200ng of pertussis toxin.

Rojas O.L., Pröbstel A.K., Porfilio E.A., Wang A.A., Charabati M., Sun T., Lee D.S., Galicia G., Ramaglia V., Ward L.A., Leung L.Y., Najafi G., Khaleghi K., Garcillán B., Li A., Besla R., Naouar I., Cao E.Y., Chiaranunt P., Burrows K., Robinson H.G., Allanach J.R., Yam J., Luck H., Campbell D.J., Allman D., Brooks D.G., Tomura M., Baumann R., Zamvil S.S., Bar-Or A., Horwitz M.S., Winer D.A., Mortha A., Mackay F., Prat A., Osborne L.C., Robbins C., Baranzini S.E, & Gommerman J.L. (2019). Recirculating Intestinal IgA-Producing Cells Regulate Neuroinflammation via IL-10. Cell, 176(3), 610-624.e18.

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Experimental Autoimmune Encephalomyelitis Model

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For active EAE, mice were immunized subcutaneously (s.c.) on the back with 200 μg of MOG_35-55 (MEVGWYRSPFSRVVHLYRNGK) emulsified in CFA (Difco Lab, Detroit, MI) containing 4 mg/ml Mycobacterium tuberculosis H37Ra (Difco). Two hundred ng of pertussis toxin (List Biological Lab, Epsom, England) was given i.p. on days 0 and 2 post immunization (p.i.). For passive EAE, GFAP-shIFN-γR or GFAP-shVec lentivirus injected mice were transferred with 3.0×10⁷ polarized MOG_35-55-specific Th1 or Th17 cells/mouse 4 hours after sublethal irradiation (550 Rad). To prepare MOG-specific polarized T cell populations, draining lymph nodes and spleen cells were prepared from mice immunized as described above at day 9 p.i. Cells were cultured for 4 days with MOG_35-55 at a concentration of 25 μg/ml under Th1- (20 ng/ml rmIL-12 [PeproTech], 2 μg/ml anti-IL23p19 [eBioscience]) or Th17- (20 ng/ml rmIL-23 [PeproTech]) polarizing conditions (28 (link)). Mice were scored daily for appearance of clinical signs of EAE on a scale from 0 to 5 as described previously (29 (link)): 0, no clinical signs; 1, fully limp tail; 2, paralysis of one hind limb; 3, paralysis of both hind limbs; 4, paralysis of trunk; 5, moribund or death.

Ding X., Yan Y., Li X., Li K., Ciric B., Yang J., Zhang Y., Wu S., Xu H., Chen W., Lovett-Racke A.E., Zhang G.X, & Rostami A. (2015). Silencing IFN-γ binding/signaling in astrocytes vs. microglia leads to opposite effects on CNS autoimmunity. Journal of immunology (Baltimore, Md. : 1950), 194(9), 4251-4264.

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T-cell-transfer Experimental Autoimmune Encephalomyelitis

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Six-week-old C57BL/6 female mice were purchased from Orient Bio (Gyeonggi, Korea). For T-cell-transfer EAE, mice were immunized with 300 μg myelin oligodendrocyte glycoprotein peptide (MOG_35–55) peptide emulsified in complete freund’s adjuvant (CFA) with heat-inactivated Mycobacterium tuberculosis. Seven days after immunization, cells from draining lymph nodes were isolated and cultured with 20 μg/mL MOG_35–55 and IL-23 (20 ng/mL) (PeproTech, Rocky Hill, NJ, USA). On day five post in vitro stimulation, cells were harvested and enriched with CD4⁺ T cells by using magnetic beads. The enriched CD4⁺ T cells (2 × 10⁶ cells/mouse) were transferred into mice followed by immunization with MOG_35–55 in CFA (s.c.) and subsequent pertussis toxin (PT) (i.p.) injection, and the clinical severity for EAE pathology was monitored daily as previously mentioned [20 (link)].

Park Y.J., Cho M., Choi G., Na H, & Chung Y. (2020). A Critical Regulation of Th17 Cell Responses and Autoimmune Neuro-Inflammation by Ginsenoside Rg3. Biomolecules, 10(1), 122.

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Adoptive Transfer of Autoimmune Encephalomyelitis

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Donor mice (Cre⁻ littermates of Pdgfrb-CreERT2-MHCII mice) were immunized with 50 µg MOG_35–55 peptide (generated by Rudolf Volkmer, Charite Berlin, Germany) emulsified in complete Freund’s adjuvant (Difco by BD, Sparks, MD, USA; 263810) including Mycobacterium tuberculosis (Difco by BD, Sparks, MD, USA; 231141) by s.c. injection. After 10 days immune cells from lymph nodes and spleen were isolated and in vitro re-stimulated for 72 h in the presence of 20 mg/mL MOG_35–55 and 20 ng/mL IL-12 (Peprotech, Hamburg, Germany; 210-12). Cells (5 × 10⁶) were injected i.p. into tamoxifen-treated recipient mice (Cre⁻ mice = MHC II WT, Cre⁺ mice = MHC II KO).

Koch K., Lindner M., Fleck A.K., Liebmann M., Eschborn M., Zondler L., Diéguez-Hurtado R., Adams R.H., Meyer zu Hörste G., Zarbock A., Kuhlmann T., Wiendl H, & Klotz L. (2022). CNS Pericytes Modulate Local T Cell Infiltration in EAE. International Journal of Molecular Sciences, 23(21), 13081.

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