Cd4 cd25 cd127dim regulatory t cell isolation kit

Manufactured by Miltenyi Biotec

Sourced in Germany, United States

The CD4+CD25+CD127dim/− regulatory T cell isolation kit is a laboratory equipment product designed to isolate CD4+CD25+CD127dim/− regulatory T cells from human or murine samples. The kit utilizes magnetic bead-based separation technology to enrich for this specific cell population.

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Lab products found in correlation

Ficoll, by Solarbio (1 mentions) Interleukin 2 (il 2), by BioLegend (1 mentions) Anti cd3, by BioLegend (1 mentions) Anti cd28, by BioLegend (1 mentions) Horizon violet proliferation dye 450, by BD (1 mentions)

Close spelling variants of this product

CD4+CD25+CD127dim/– Regulatory T Cells Isolation Kit II CD4+CD25+ isolation kits Cell isolation kits Isolation kits

3 protocols using cd4 cd25 cd127dim regulatory t cell isolation kit

Tregs Isolation, Activation, and Coculture with Tumor Cells

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Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (Solarbio, Cat#P8900) density gradient centrifugation. Tregs were purified from PBMCs with the CD4⁺CD25⁺CD127^dim/− regulatory T cell isolation kit (Miltenyi Biotec, Cat#130-094-775) according to the manufacturer’s instructions. The isolated Tregs were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (FBS), 100 U/mL interleukin 2 (BioLegend, Cat#589106), 1 µg/µl anti-CD3 (BioLegend, Cat#300314), and 1 µg/µl anti-CD28 (BioLegend, Cat#302934). After stimulation for 3 days, Tregs were cocultured with tumor cells at a 5:1 ratio for 48 hours in a transwell system with 0.4 µm pores (Corning, Cat#3413). Then, Tregs were collected and stained for apoptosis and programmed cell death-1 (PD-1) analysis.

Wei J.L., Wu S.Y., Yang Y.S., Xiao Y., Jin X., Xu X.E., Hu X., Li D.Q., Jiang Y.Z, & Shao Z.M. (2021). GCH1 induces immunosuppression through metabolic reprogramming and IDO1 upregulation in triple-negative breast cancer. Journal for Immunotherapy of Cancer, 9(7), e002383.

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Quantifying Treg Suppressive Function in CRC

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To assess the suppressive properties of Tregs isolated from the peripheral blood of CRC patients, we tested their ability to inhibit the proliferation of T effector cells (Teff), as previously described (36 (link)). Briefly, peripheral Tregs were magnetically separated from CRC patients’ PBMC, using the CD4⁺CD25⁺CD127^dim/− Regulatory T Cell Isolation Kit (Miltenyi Biotec, Germany): cells eluted from the column constituted Tregs, whereas T cells not retained by the magnetic field were accounted as Teff. Tregs and Teff were cultured alone or together at a 1:1 ratio in complete medium, with or without polyclonal stimulation [1 µg/ml, low-endotoxin anti-CD3 and anti-CD28 monoclonal antibodies (BD Biosciences)]. The stimulation was carried out immediately after BD Horizon Violet Proliferation Dye 450 (VPD450, BD Biosciences) incorporation. The proliferation of Teff, alone or in the presence of Tregs, was assessed after 5-day incubation by flow cytometry.

Niccolai E., Ricci F., Russo E., Nannini G., Emmi G., Taddei A., Ringressi M.N., Melli F., Miloeva M., Cianchi F., Bechi P., Prisco D, & Amedei A. (2017). The Different Functional Distribution of “Not Effector” T Cells (Treg/Tnull) in Colorectal Cancer. Frontiers in Immunology, 8, 1900.

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Isolation of Regulatory T-cells from PBMCs

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Using CD4⁺CD25⁺CD127^dim regulatory T-cell isolation kit (Miltenyi Biotec Inc, Auburn, CA, USA), naturally occurring Tregs were isolated from adult PB as per manufacturer’s instructions.

Liu X., Robinson S., Setoyama T., Tung S., D’Abundo L., Shah M., Yang H., Yvon E., Shah N., Yang H., Konopleva M., Garcia-Manero G., McNiece I., Rezvani K., Calin G., Shpall E, & Parmar S. (2014). FOXP3 is a direct target of miR15a/16 in umbilical cord blood regulatory T cells. Bone marrow transplantation, 49(6), 793-799.

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