Cd4 cd25 treg isolation kit

Manufactured by Miltenyi Biotec

Sourced in Germany

The CD4+CD25+ Treg Isolation Kit is a laboratory equipment product designed for the isolation of CD4+CD25+ regulatory T cells (Tregs) from human or mouse samples. The kit utilizes a magnetic bead-based separation method to selectively isolate the target cell population.

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Lab products found in correlation

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CD4+CD25+ isolation kits Treg isolation kit Isolation kits

15 protocols using cd4 cd25 treg isolation kit

In vitro Treg Cell Differentiation and Polarization

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In vitro induced Treg (iT_reg) cells were differentiated as previously described (15 (link)). Briefly, CD4⁺CD25⁻ cells were negatively selected from B6 or Pbx1-d Tg splenocytes using the CD4⁺CD25⁺ Treg isolation kit (Miltenyi Biotech), and then 5 × 10⁵ cells were stimulated with mouse T-activator CD3/CD28 beads (Life Technologies) at the concentration of 1 × 10⁶ beads/mL in the presence of 100 U IL-2, 20 ng/ml TGF-β (Pepro Tech), and 0–5 nM all-trans retinoic acid (RA) for 5 d. T_H1 and T_H17 polarization was performed as previously described (23 ).

Choi S.C., Hutchinson T.E., Titov A.A., Seay H.R., Li S., Brusko T.M., Croker B.P., Salek-Ardakani S, & Morel L. (2016). The lupus susceptibility gene Pbx1 regulates the balance between follicular helper T cell and regulatory T cell differentiation. Journal of immunology (Baltimore, Md. : 1950), 197(2), 458-469.

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Isolation of Human Regulatory T Cells

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Peripheral blood mononuclear cells (PBMC) were isolated from freshly collected blood of healthy volunteers or from buffy coats by Ficoll density-gradient centrifugation (PAA Laboratories, Pasching, Austria). Treg isolation was performed using the CD4+CD25+ Treg isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s recommendations. This study was approved by the Charité-Universitätsmedizin Berlin ethics committee (Institutional Review Board) and all blood donors provided written informed consent.

Landwehr-Kenzel S., Zobel A., Schmitt-Knosalla I., Forke A., Hoffmann H., Schmueck-Henneresse M., Klopfleisch R., Volk H.D, & Reinke P. (2021). Cyclosporine A but Not Corticosteroids Support Efficacy of Ex Vivo Expanded, Adoptively Transferred Human Tregs in GvHD. Frontiers in Immunology, 12, 716629.

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Isolation of CD4+CD25+ Regulatory T Cells

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From pooled splenocytes, CD4⁺CD25⁺ cells were purified using CD4⁺CD25⁺ Treg isolation kit (Miltenyi Biotec Inc., Auburn, CA) according to the manufacturer's protocol. CD4⁺CD25^- cells were obtained by collecting the flow through cells during positive selection of CD4⁺CD25⁺ cells. Confirmation of >80% purity was performed by FACS before in vitro culture.

Sarikonda G., Fousteri G., Sachithanantham S., Miller J.F., Dave A., Juntti T., Coppieters K.T, & von Herrath M. (2014). BDC12-4.1 T-Cell Receptor Transgenic Insulin-Specific CD4 T Cells Are Resistant to In Vitro Differentiation into Functional Foxp3+ T Regulatory Cells. PLoS ONE, 9(11), e112242.

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Isolation of Human Peripheral Tregs

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Written informed consent to participate in the study was obtained from the patients and donors before blood sampling. Blood samples were obtained via venipuncture into vacuum tubes with K₂EDTA anticoagulant. Density gradient centrifugation using 1.077 g/mL Ficoll (Paneco, Moscow, Russia) was used to obtain peripheral blood mononuclear cells. Then, Tregs were isolated from peripheral blood mononuclear cells by immune-magnetic separation using the CD4+CD25+Treg isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany).

Zhdanov D.D., Gladilina Y.A., Blinova V.G., Abramova A.A., Shishparenok A.N, & Eliseeva D.D. (2024). Induction of FoxP3 Pre-mRNA Alternative Splicing to Enhance the Suppressive Activity of Regulatory T Cells from Amyotrophic Lateral Sclerosis Patients. Biomedicines, 12(5), 1022.

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Adoptive Transfer of T Cell Subsets

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Splenocytes from WT, PD-1 KO, T-bet Tg and P/T mice were isolated and single-cell suspensions were prepared in PBS. Splenic cells (1·0 × 10⁷) were injected intravenously into the recipient adult Rag-2 KO mice. In other experiments, the CD4⁺CD25⁺ T_regs were isolated from splenocytes of WT mice using CD4⁺CD25⁺ T_reg isolation kit (Miltenyi Biotec). The purity of all populations was determined with FACS before injection. Mice were observed every other day for clinical signs of autoimmune syndromes. Histological determination of autoimmune responses against various organs and tissues was conducted at 10 weeks after transfer.

Tahara M., Kondo Y., Yokosawa M., Tsuboi H., Takahashi S., Shibayama S., Matsumoto I, & Sumida T. (2015). T-bet regulates differentiation of forkhead box protein 3+ regulatory T cells in programmed cell death-1-deficient mice. Clinical and Experimental Immunology, 179(2), 197-209.

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CD4+ CD25+ T Cell Isolation and Adoptive Transfer

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T cells were purified from spleens of WT and Gpr174 KO mice, mechanically disrupted over a 75 μm filter, and subjected to magnetic bead enrichment for CD4⁺CD25⁺ T cells using CD4⁺CD25⁺ Treg isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Enriched CD4⁺CD25⁺ T cells were counted and resuspended in PBS. A cell suspension containing 1 × 10⁶ per 100 μl PBS was injected into C57BL/6 Rag2^−/− mouse via tail vein.

Qiu D., Chu X., Hua L., Yang Y., Li K., Han Y., Yin J., Zhu M., Mu S., Sun Z., Tong C, & Song Z. (2019). Gpr174-deficient regulatory T cells decrease cytokine storm in septic mice. Cell Death & Disease, 10(3), 233.

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In Vitro Suppression Assay of Regulatory T Cells

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CD4⁺ CD25⁻effector T cells and CD4⁺ CD25⁺ Tregs were isolated from B6 or B6.SLE123 spleens by using a CD4⁺ CD25⁺ Treg isolation kit (Miltenyi, San Diego, CA). Effector CD4 T cells and CD4Tregs were resuspended in complete RPMI media (10% fetal calf serum plus 1% penicillin/streptomycin) and plated in 96-well round-bottomed plates at Treg:T effector ratios ranging from 1:2 to 1:8 (8 (link)). Cells were stimulated with anti-CD3 (1 µg/mL, 145-2C11; BD) and anti-CD28 (1 µg/mL, 37.51; BD) and incubated for 72 h. For transforming growth factor-β (TGF-β) suppression assays, splenocytes were incubated with anti-CD3/CD28 (1 µg/mL) with or without 10 µg/mL TGF-β (R&D Systems, Minneapolis, MN) for 72 h. Supernatants were collected and analyzed for interferon-γ (IFNγ) by enzyme-linked immunosorbent assay (ELISA) (BD). The percent suppression was calculated as follows: [(IFNγ production in each condition/IFNγ T effector cells only) * 100].

Stocks B.T., Wilhelm A.J., Wilson C.S., Marshall A.F., Putnam N.E., Major A.S, & Moore D.J. (2015). Lupus-Prone Mice Resist Immune Regulation and Transplant Tolerance Induction. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 16(1), 334-341.

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T Cell Proliferation and Differentiation Assay

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As presented in Supplementary figure 1, Treg (CD4⁺CD25^high) and effector T cells (CD4⁺CD25^low; Teff) were purified from fresh PBMCs by magnetic cell sorting following the manufacturers' instructions (CD4⁺CD25⁺ Treg Isolation Kit; Miltenyi Biotec, Paris, France). Teff and Treg were labelled with CellTrace Violet and CFSE (Thermo Fisher Scientific, Invitrogen, VILLEBON‐SUR‐YVETTE, France), respectively. T cells were activated with anti‐CD2/CD3/CD28 microbeads (Miltenyi Biotec) and cultured with or without Treg (Teff:Treg ratio = 2:1) or tocilizumab (5 µg mL⁻¹). After 4 days, cells were activated with 0.1 µg mL⁻¹ of phorbol 12‐myristate 13‐acetate (PMA) and 1 µg mL⁻¹ of ionomycin (Sigma‐Aldrich, Saint‐Quentin‐Fallavier, France) in the presence of Brefeldin A (BD Golgi Plug; BD Bioscience, Le Pont‐de‐Claix, France) for 4 h and then fixed, permeabilised and stained with CD4‐PECy7, IL‐17‐PE and IFN‐γ‐APC. CellTrace Violet or CFSE incorporation and cytokine expression were analysed by flow cytometry to determine the proliferation index of Teff and Treg and the percentage of Th1 (CD4⁺IFN‐γ⁺) and Th17 (CD4⁺IL‐17⁺) cells among Teff (CD4⁺CFSE⁻) and Treg (CD4⁺CFSE⁺).

Samson M., Greigert H., Ciudad M., Gerard C., Ghesquière T., Trad M., Corbera‐Bellalta M., Genet C., Ouandji S., Cladière C., Thebault M., Ly K.H., Liozon E., Maurier F., Bienvenu B., Terrier B., Guillevin L., Charles P., Quipourt V., Devilliers H., Gabrielle P., Creuzot‐Garcher C., Tarris G., Martin L., Saas P., Audia S., Cid M.C, & Bonnotte B. (2021). Improvement of Treg immune response after treatment with tocilizumab in giant cell arteritis. Clinical & Translational Immunology, 10(9), e1332.

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Intracellular Transcription Factor Staining

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For Foxp3, cells were stained with anti-Foxp3 (eBioscience) using a Foxp3 staining kit (BioLegend). For YY1 staining, total splenocytes from YY1-eGFP mice were enriched by CD4 microbeads (Miltenyi). T_conv (CD4⁺CD25⁻) or T_reg (CD4⁺ CD25⁺) cells from WT mice were prepared using a CD4⁺CD25⁺ T_reg isolation kit (Miltenyi). The cells were stained with an anti-YY1 (Santa Cruz sc-1703) antibody using a Foxp3 staining kit (BioLegend). For cytokine staining, cells were restimulated with 1 μM ionomycin (Sigma-Aldrich) and 10 nM phorbol myristate acetate (Sigma-Aldrich) with Golgi stop (BD Bioscience) for 4 h. Intracellular staining was performed using Cytofix/cytoperm kit (BD Bioscience).

Hwang S.S., Jang S.W., Kim M.K., Kim L.K., Kim B.S., Kim H.S., Kim K., Lee W., Flavell R.A, & Lee G.R. (2016). YY1 inhibits differentiation and function of regulatory T cells by blocking Foxp3 expression and activity. Nature Communications, 7, 10789.

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Isolation of Immune Cell Subsets

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CD4⁺CD25⁺ Tregs and CD4⁺CD25⁻ responder T cells (Tresp) were isolated from lymph nodes of naïve BALB/c mice by magnetic assisted cell sorting using the CD4⁺CD25⁺ Treg Isolation Kit (Miltenyi Biotec, Bergisch-Gladbach, Germany). NK cells were sorted from draining lymph nodes and splenocytes of BALB/c mice using the NK Cell Isolation Kit II (Miltenyi Biotec). Purity of isolated Tregs was >90% as measured by flow cytometry. Antigen presenting cells (APCs) were obtained by depleting T cells (CD90.2⁺ cells) from splenocytes of BALB/c mice using a CD90.2 isolation kit (Miltenyi Biotec).

Nakao T., Inomata T., Blanco T., Musayeva A., Tahvildari M., Amouzegar A., Yin J., Chauhan S.K., Chen Y, & Dana R. (2022). Amplified Natural Killer Cell Activity and Attenuated Regulatory T Cell Function Are Determinants for Corneal Alloimmunity in the Very Young Mice. Transplantation, 107(6), 1302-1310.

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