Cd25 microbeads 2

Manufactured by Miltenyi Biotec

Sourced in United Kingdom, Germany

CD25 MicroBeads II are magnetic beads coated with antibodies specific for the CD25 antigen. They are used for the isolation and enrichment of CD25-positive cells from heterogeneous cell populations.

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Lab products found in correlation

Cd4 t cell isolation kit, by Miltenyi Biotec (5 mentions) Anti hla dr microbeads, by Miltenyi Biotec (4 mentions) Rosettesep human cd4 t cell enrichment cocktail, by STEMCELL (3 mentions) Ficoll hypaque gradient, by GE Healthcare (3 mentions) Lymphoprep, by Axis-Shield (2 mentions) Cd4 t cell isolation kit 2, by Miltenyi Biotec (2 mentions) Automacs, by Miltenyi Biotec (2 mentions) Cd69 microbead kit 2, by Miltenyi Biotec (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Facscanto 2, by FlowJo (1 mentions) Cd25 pe cy7, by BD (1 mentions) Cd127 percp cy5, by BioLegend (1 mentions) Lithium heparin coated tubes, by Greiner (1 mentions) Facsaria 2 cell sorter, by BD (1 mentions) Ls column, by Miltenyi Biotec (1 mentions) Ficoll paque plus, by Merck Group (1 mentions) Anti cd4 apc cy7, by BD (1 mentions) Hil 7r m21, by BD (1 mentions) Mem 56, by Thermo Fisher Scientific (1 mentions) Rpa t4, by BioLegend (1 mentions)

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CD25 microbeads (37 citations)

18 protocols using cd25 microbeads 2

Isolation of Regulatory T Cell Subsets

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Human buffy coats (New York Blood Center) were first incubated with RosetteSep™ Human CD4⁺CD127^low T Cell Enrichment Cocktail (Stemcell Technologies, Catalog #15361). The cells isolated from the Ficoll interface were then incubated with CD25 MicroBeads II (Miltenyi Biotec, Catalog #130–092-983) and underwent positive selection by the autoMACS Pro Separator. The purity of the CD4⁺CD127⁻CD25⁺ and CD25⁻ populations was confirmed by flow cytometry staining for the extracellular markers CD3 (Biolegend, Catalog #317306, CloneOKT3), CD4 (Invitrogen, Catalog #MHCD0412, Clone S3.5), and CD25 (Miltenyi Biotec, Catalog #130–0920-858, Clone 4E3) and the intracellular markers Foxp3 (eBioscience, Catalog #48–4777-42, Clone 236A/E7) and Helios (Biolegend, Catalog #137,216, Clone 22F6).

Weingartner E., Courneya J.P., Keegan A, & Golding A. (2016). A novel method for assaying human regulatory T cell direct suppression of B cell effector function. Journal of immunological methods, 441, 1-7.

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CD4+ T Cell Isolation from PBMC

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Peripheral blood was obtained from anonymized leukocyte cones supplied by the National Blood Transfusion Service (NHS Blood and Transplantation, Tooting, London, UK). Peripheral blood mononuclear cells (PBMC) were isolated by lympholyte (1.077 g/cm³) gradient stratification (Lymphoprep, Axis-Shield, Oslo, Norway). RosetteSep Human CD4+ T cell enrichment cocktail (STEMCELL Technologies, Vancouver, BC, Canada) was used to purify CD4+ T cell fraction, and CD25 Microbeads II (Miltenyi Biotec, Surrey, UK) were used to separate CD4+CD25− T cells from CD4+CD25+ T cell fraction according to manufacturer’s instructions.

Fanelli G., Romano M., Lombardi G, & Sacks S.H. (2023). Soluble Collectin 11 (CL-11) Acts as an Immunosuppressive Molecule Potentially Used by Stem Cell-Derived Retinal Epithelial Cells to Modulate T Cell Response. Cells, 12(13), 1805.

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Assessing Teff Resistance to Treg Suppression

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Teff resistance to suppression was measured using an in vitro Treg-mediated suppression assay using Teff cell surface expression of CD25 and CD134 as a surrogate marker of Treg-mediated suppression (6 (link), 29 (link)). In brief, CD4⁺ T cells depleted of CD25^hi cells were isolated from PBMCs using a no-touch Miltenyi CD4 T Cell Isolation Kit II and positive Miltenyi CD25 microbeads II. CD4⁺CD25⁺CD127^lo Tregs from a single healthy donor were sorted, expanded, and frozen as described (47 (link)) and used a constant source of Tregs for all suppression assays. CD4⁺CD25^dim Teffs were cultured at 100,000 cells per well. Tregs were added at ratios of 1:4, 1:8, 1:16, and 1:32 (Treg/Teff), and Dynabeads CD3/CD28 T Cell Expander beads (Life Technologies) were added at a ratio of 1:28 (beads/Teffs). On day 2, Teffs were stained with CD25 and CD134 (Supplemental Table 2). For analysis, Teffs cultured in media alone were used to set gates for the various activation markers or proliferation. EF670 was used to identify Tregs. Percentage suppression (s) was calculated as follows: s = ([a − b]/a) × 100, where a is the percentage of CD25⁺CD134⁺ Teffs in the absence of Tregs and b is the percentage of CD25⁺CD134⁺ Teff cells in the presence of Tregs. Samples were collected on a BD Biosciences FACSCanto II, and data were analyzed using FlowJo V10.6.2.

Speake C., Habib T., Lambert K., Hundhausen C., Lord S., Dufort M.J., Skinner S.O., Hu A., Kinsman M., Jones B.E., Maerz M.D., Tatum M., Hocking A.M., Nepom G.T., Greenbaum C.J, & Buckner J.H. (2022). IL-6–targeted therapies to block the cytokine or its receptor drive distinct alterations in T cell function. JCI Insight, 7(22), e159436.

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Isolation and Purification of Human Tregs and Tconvs

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The study was conducted according to the Declaration of Helsinki principles and was approved by the institutional ethics committee of Hasselt University (Comité voor Medische Ethiek UHasselt, CME2019/042 and CME2016/629). Peripheral blood (collected in Lithium Heparin coated tubes, 455084, Greiner bio-one) or buffy coats (purchased from the Belgium Red Cross) were obtained from healthy donors, after providing written informed consent. Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque PLUS (GE17-1440-02, Sigma-Aldrich) gradient centrifugation. In some cases, CD4⁺ T cells were isolated from whole blood using RosetteSep™ Human CD4⁺ T Cell Enrichment Cocktail (15062, Stemcell Technologies) according to manufacturer’s protocol. CD4⁺ T cells or PBMCs were then incubated with CD25 Microbeads II (130-097-044, Miltenyi Biotec) and separated using LS columns (Miltenyi Biotec). Tregs were isolated from CD25⁺ enriched cells, while Tconvs were isolated from CD25-depleted cells. Cells were labelled with Propidium Iodide (PI) (556463, BD Biosciences) prior being sorted as PI^-CD4⁺CD25⁺CD127^- Tregs or as PI^-CD4⁺CD25^-CD127⁺ Tconvs by FACS using a BD FACSAria II cell sorter and anti-CD4 APC-Cy7 (clone RPA-T4, 557851, BD Biosciences), CD25 PE-Cy7 (clone M-A251, 557741, BD Biosciences) and CD127 PerCP-Cy5.5 (clone A019D5, 351322, Biolegend) antibodies.

Côrte-Real B.F., Arroyo Hornero R., Dyczko A., Hamad I, & Kleinewietfeld M. (2022). Dissecting the role of CSF2RB expression in human regulatory T cells. Frontiers in Immunology, 13, 1005965.

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Isolation and Expansion of Human nTregs

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Human natural Tregs (nTregs: CD4+CD25highCD127lowCD45RA+) were isolated from human peripheral blood mononuclear cells (PBMCs) after Ficoll (GE Healthcare) gradient separation and CD25 preenrichment (CD25 MicroBeads II, MiltenyiBiotech) via AutoMACS (MiltenyiBiotech). For fluorescence-activated cell scanning (FACS), PBMCs were labelled with monoclonal antibody combinations: CD4+ (RPA-T4, BioLegend), CD25+ (2A3, BD), CD127- (hIL-7R-M21, BD), and CD45RA+ (MEM-56, Thermo Fisher). HLA-A*02 status was confirmed by flow cytometry via α-HLA-A2/A28 (REA 142, Miltenyi Biotec). FACS-based cell sorting was performed at the cell-sorting facility of Hannover Medical School. The obtained purity of isolated Treg cells was >95%. Tregs were kept in TexMacs GMP cell culture medium (Miltenyi Biotech) supplemented with 10% human AB serum, 1% penicillin−streptomycin (Gibco), 1 mM sodium pyruvate, 1% nonessential amino acids (NEAA, Gibco), 20 mM HEPES and 50 µM beta-mercaptoethanol with the indicated concentrations of IL-2 (Proleukin, Clinigen). Tregs were expanded by using Treg expansion beads (MiltenyiBiotech) according to the manufacturer’s instructions.

Kremer J., Henschel P., Simon D., Riet T., Falk C., Hardtke-Wolenski M., Wedemeyer H., Noyan F, & Jaeckel E. (2022). Membrane-bound IL-2 improves the expansion, survival, and phenotype of CAR Tregs and confers resistance to calcineurin inhibitors. Frontiers in Immunology, 13, 1005582.

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Isolation and Expansion of Human Regulatory T Cells

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Anonymous healthy donor blood was obtained from the National Blood Service (NHS Blood and Transplantation, London, United Kingdom) with informed consent and ethical approval (Institutional Review Board of Guy’s Hospital; reference 09/H0707/86). RosetteSep^TM (StemCell Technologies, Cambridgeshire, United Kingdom) was used to select CD4⁺ T cells. Subsequently, CD25 Microbeads II (Miltenyi Biotec, Surrey, United Kingdom) were used to separate CD4⁺CD25⁺ (Tregs, Supplementary Figures S1A,B,E) and CD4⁺CD25^– (Teffs, Supplementary Figures S1C,D) cells. Tregs were cultured and expanded for 20 days as previously reported (Scottà et al., 2013 (link)). Briefly, isolated Tregs were cultured in X-VIVO 15 medium (Lonza, Berkshire, United Kingdom) supplemented with 5% heat-inactivated human AB serum (Biosera, Sussex, United Kingdom) in the presence of anti-CD3/CD28-coated beads (Thermo Fisher Scientific, Paisley, United Kingdom), 100 nM rapamycin (LC-Laboratories, MA, United States) and 1000 U/mL recombinant IL-2 (Proleukin-Novartis, Camberley, Surrey, United Kingdom). Teffs were cultured in the same media in the presence of anti-CD3/CD28-coated beads and 100 U/mL recombinant IL-2. All cell cultures were tested for mycoplasma and all samples used were mycoplasma free.

Tung S.L., Fanelli G., Matthews R.I., Bazoer J., Letizia M., Vizcay-Barrena G., Faruqu F.N., Philippeos C., Hannen R., Al-Jamal K.T., Lombardi G, & Smyth L.A. (2020). Regulatory T Cell Extracellular Vesicles Modify T-Effector Cell Cytokine Production and Protect Against Human Skin Allograft Damage. Frontiers in Cell and Developmental Biology, 8, 317.

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PBMC Isolation and CD4+ T Cell Purification

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Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors by a Ficoll-Hypaque gradient (GE Healthcare, Uppsala, Sweden). Human rCD4 T lymphocytes were isolated with a CD4 T Cell Isolation Kit (Miltenyi Biotec, Cologne, Germany) and the subsequent depletion of CD25⁺ and HLA-DR⁺ cells was performed using CD25 MicroBeads II and anti-HLA-DR MicroBeads (Miltenyi Biotec), respectively.

López-Huertas M.R., Morín M., Madrid-Elena N., Gutiérrez C., Jiménez-Tormo L., Santoyo J., Sanz-Rodríguez F., Moreno Pelayo M.Á., Bermejo L.G, & Moreno S. (2019). Selective miRNA Modulation Fails to Activate HIV Replication in In Vitro Latency Models. Molecular Therapy. Nucleic Acids, 17, 323-336.

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Dual-reporter Luciferase Assay for JMJD8 Activity

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A dual-reporter luciferase assay (Promega) was performed in HEK293T cells in which thymidine-kinase-promoter-driven Renilla luciferase vector (pRL-TK) was used as the reporter and the pro-moterless firefly luciferase vector (pGL3-Basic) was used as the normalizing control at a 1:5 ratio. To test the activity of JMJD8 and ΔN-JMJD8, 1 μg of the corresponding overexpression construct (pEF1-JMJD8-V5/His or pEF1-ΔN-JMJD8-V5/His) or control (pEF1-mock) was cotransfected into the cells using Mirus TransIT-293T transfection reagent (Mirus Bio). In a second approach, we performed a single-reporter assay using SV40-promoter-driven luciferase plasmid (pGL3-SV40P) transfected together with pEF1 vector coexpressing JMJD8 with a human CD25 surface antigen (pEF1-FH-JMJD8-IRES-CD25) or mock (pEF1-IRES-CD25) into HEK293T cells. Forty-eight hours after transfection, cells were sorted using microbeads conjugated to mono-clonal anti-human CD25 antibodies (CD25 Microbeads II, Miltenyi Biotec) on an MS column (Myltenyl Bio). Luciferase assays were performed on the CD25-enriched populations; readings were further normalized to the total protein content of the cells determined by Bradford assay.

Khoueiry R., Sohni A., Thienpont B., Luo X., Velde J.V., Bartoccetti M., Boeckx B., Zwijsen A., Rao A., Lambrechts D, & Koh K.P. (2017). Lineage-specific functions of TET1 in the postimplantation mouse embryo. Nature genetics, 49(7), 1061-1072.

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Isolation and Expansion of CD4+ Treg Cells

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We obtained peripheral blood samples from 3 healthy donors and 3 prostate cancer patients. PBMCs were isolated by density gradient centrifugation with Ficoll-Paque (GE Healthcare). Human primary CD4+ CD25-T cells or CD4+ CD25+ Treg cells were purified using the CD4+ T Cell Isolation Kit (Miltenyi Biotec) and CD25 MicroBeads II (Miltenyi Biotec) (Figure S1). CD4+ CD25+ Treg cells were cultured with plate-bound anti-human-CD3 (OKT3; eBiosciences) antibodies (5 μg ml−1) and/or anti-human-CD28 (CD28.2; BD Pharmingen) antibodies (2 μg ml−1) in complete medium [RPMI supplemented with 10% FBS and IL-2 (Peprotech) (100 units ml−1)]. Then, CD4+ CD25+ CD127- Treg cells were enriched using flow cytometry (Figure S1). All cells were cultured at 37°C in an atmosphere of 5% CO2.

Ju M., Fan J., Zou Y., Yu M., Jiang L., Wei Q., Bi J., Hu B., Guan Q., Song X., Dong M., Wang L., Yu L., Wang Y., Kang H., Xin W, & Zhao L. (2022). Computational Recognition of a Regulatory T-cell-specific Signature With Potential Implications in Prognosis, Immunotherapy, and Therapeutic Resistance of Prostate Cancer. Frontiers in Immunology, 13, 807840.

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Isolation of Resting CD4+ T Cells from PBMCs

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Bcl2 model cells were generated as previously described [53 (link)]. Peripheral blood mononuclear cells (PBMCs) from HIV negative and positive study participants were purified on Ficoll-paque PLUS (GE Healthcare, Cat#17-1440-02). CD4⁺ T lymphocytes were extracted (Miltenyi Biotec Cat#130-096-533) by negative selection. Resting CD4⁺ T cells were isolated by subsequent negative selection using CD25 MicroBeads II, CD69 MicroBead Kit II, and Anti-HLA-DR MicroBeads (Miltenyi Biotec Cat#130-092-983, Cat#130-092-355, Cat#130-046-101). Cells were kept in RPMI 1640 medium (Hyclone, Cat# SH30096_01), 10% FBS (Life Technologies, Cat# 10270–106), 1% Glutamax (Life Technologies, Cat# 35050), 1% Penicillin-streptomycin (Life Technologies, Cat# 15140–122). For active growth conditions, media was supplemented with human interleukin-2 IS (Miltenyi Biotec, Cat#130-097-742; Lot#5170516373) final concentration 100U/ml and 5% T-cell conditioned media, according to the protocol.

Lindqvist B., Svensson Akusjärvi S., Sönnerborg A., Dimitriou M, & Svensson J.P. (2020). Chromatin maturation of the HIV-1 provirus in primary resting CD4+ T cells. PLoS Pathogens, 16(1), e1008264.

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