Cd25 microbead kit

Manufactured by Miltenyi Biotec

Sourced in Germany

The CD25 MicroBead Kit is a laboratory product designed for the isolation and enrichment of CD25-positive cells from various sample types. It utilizes magnetic beads coated with antibodies specific to the CD25 cell surface marker to facilitate the separation of these cells from the rest of the sample.

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CD25 microbeads (37 citations) MicroBead kits (12 citations) Mouse CD25 MicroBead Kit (3 citations) CD25 microBeads kit (2 citations)

18 protocols using cd25 microbead kit

Isolation and Labeling of Naive T Cells

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For two-photon imaging, naïve CD4 T cells were purified by two rounds of MACS negative selection using the naïve CD4⁺ T cell isolation kit (Miltenyi Biotech) to remove non-CD4 and CD44⁺ T cells. The CD25⁺ cells were further removed from the flow-through portion using the CD25 microbead kit (Miltenyi Biotech). The naive 8.3 or WT CD8 T cells were purified similarly by using the naïve CD8⁺ T cell isolation kit. Flow Cytometry analysis confirmed that >95% of the cells were CD4⁺/CD8⁺CD25⁻CD62L^hiCD44⁻. CFSE or CMTMR labelling was performed using the Vybrant CFDA SE Cell Tracer Kit and the CellTracker Orange CMTMR Dye (both from ThermoFisher Scientific), respectively. In brief, T cells (10⁷/ml in PBS) were incubated with 10 μM CFSE or 8 μM CMTMR for 25 min at 37°C with a gentle shake after 10 min. Under these conditions, T cells were labelled with satisfactory intensities without significant cell death. Ice-cold PBS was then added to quench the labelling.

Wan X., Zinselmeyer B.H., Zakharov P.N., Vomund A.N., Taniguchi R., Santambrogio L., Anderson M.S., Lichti C.F, & Unanue E.R. (2018). Pancreatic islets communicate with lymphoid tissues via exocytosis of insulin peptides. Nature, 560(7716), 107-111.

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Isolation of Naive and Inducible Regulatory T Cell Subsets

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Naive CD4⁺ T cells from PBMC were isolated by negative selection with a naive CD4⁺ T cell isolation kit (Miltenyi Biotec, Bergisch-Gladbach, Germany). The purity of cells was ≥ 95%, as measured by flow cytometry.
CD4⁺CD25⁻Foxp3⁺ T cells were purified from TGFβ1-primed inducible CD4⁺CD25⁺Foxp3⁺ T cells with a CD25 MicroBead Kit (Miltenyi) for depletion of CD25⁺ cells, with an average purity of over 90%. For isolating inducible and peripheral CD4⁺CD25⁻CD127⁺Foxp3⁺ T cells and CD4⁺CD25⁻CD127⁻Foxp3⁺ T cells, CD4⁺CD25⁻CD45RO⁺CD127⁺ T cells, and CD4⁺CD25⁻CD45RO⁺CD127⁻ T cells were used to substitute for CD4⁺CD25⁻CD127⁺Foxp3⁺ T cells and CD4⁺CD25⁻CD127⁻Foxp3⁺ T cells, respectively. PBMC and TGFβ1-primed inducible CD4⁺CD25⁺Foxp3⁺ T cells were labeled with anti-CD4-FITC, anti-CD25-PE-Cy7, anti-CD45RO-PerCP-Cy5.5, and anti-CD127-PE. Afterwards, CD4⁺CD25⁻CD45RO⁻CD127⁺ T cells, CD4⁺CD25⁻CD45RO⁺CD127⁺ T cells, and CD4⁺CD25⁻CD45RO⁺CD127⁻ T cells were isolated by fluorescence-activated cell sorting (FACS), with an average purity >95%.

Wu J.H., Zhou M., Jin Y., Meng Z.J., Xiong X.Z., Sun S.W., Miao S.Y., Han H.L, & Tao X.N. (2019). Generation and Immune Regulation of CD4+CD25−Foxp3+ T Cells in Chronic Obstructive Pulmonary Disease. Frontiers in Immunology, 10, 220.

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Isolation and Polarization of Murine Naïve CD4+ T Cells

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Naïve (CD25-) CD4+ T cells were isolated using the CD4+ T Cell Isolation Kit, mouse and the CD25 MicroBead Kit, mouse by Miltenyi Biotec. Instead of the recommended buffer in the kit, R10 medium (RPMI-1640 with 10% FCS (Gibco) and 1% Penicillin/Streptomycin (Sigma-Aldrich)) was used and the amount of reagents suggested per 10⁷ cells was instead used per 1.5 · 10⁷ cells.
For the polarization of the CD4+ CD25- T cells, a 24-well-plate was coated with 10 μg/ml anti-CD3 and 10 μg/ml anti-CD28 in PBS for 1 h at 37 °C and washed one time with PBS before the cells were seeded at a concentration of 10⁶ cells in R10 medium for Th0 and Th1 polarization. Lastly, 5 μg/ml anti-IL-4 (BioXcell) and 10 ng/ml IL-12 (R&D systems) diluted in PBS were added for Th1 polarization. For IL-6 treatment during polarization, 10 ng/ml or 100 ng/ml IL-6 (Miltenyi Biotec) were supplemented. The cells were in total cultured for five days at 37 °C and 5% CO₂, with a medium change on day four. On day five, the cells were stimulated by addition of eBioscience™ Cell Stimulation Cocktail (plus protein transport inhibitors) (500X) (Invitrogen) for 3.5 h before harvest of supernatant for ELISA and cells for flow cytometry. The shown data after in vitro polarization are representative of at least four (Figs. 2, 3) or two (Fig. 4) independent experiments.

Matthe D.M., Thoma O.M., Sperka T., Neurath M.F, & Waldner M.J. (2022). Telomerase deficiency reflects age-associated changes in CD4+ T cells. Immunity & Ageing : I & A, 19, 16.

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Foxp3-GFP T-cell Polarization Protocol

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Single-cell suspensions were prepared from spleen and lymph nodes from 6-8-week-old C57BL/6-Tg (Foxp3-GFP) 90Pkraj/J mice (in vitro studies) or 6–8-week-old Foxp3-GFP-Balb/cJ (in vivo studies). Red blood cells were removed using 1x ACK lysis buffer (Gibco, A1049201). CD4 cells were isolated by depleting CD25⁺ cells using the CD25 MicroBead Kit (130-091-072, Miltenyi Biotec) and enriched using a CD4 T Cell Isolation Kit (130-104-454, Miltenyi Biotech). Cells were cultured in anti-mouse-CD3-coated plates in complete DMEM (MT10013CV, TFS) with 1% NEAE, 1% Penicillin-Streptomycin, 100IU/mL, L-glutamine, 10% FBS. Complete media was supplemented with β-Mercaptoethanol (21985023, TFS), 5 ng/mL of TGF-β (7666-MB-005, R&D Systems), IL-2 (402-ML-020, R&D Systems), anti-IFN-γ (BE0055, Invivogen), anti-IL-4 (BE0045, Invivogen), and 1 μg/mL anti-CD28 (16-0281-86, TFS); and cultured in 5 μg/mL anti-CD3 (16-0031-85, TFS) coated plates at a concentration of 10⁶ cells/mL. On day 3, cells were cultured in RPMI medium with 20 ng/mL IL-2. Cells were harvested on day 7 for studies. Over 90% of T_reg-polarized cells expressed CD25 and GFP.

Jorapur A., Marshall L.A., Jacobson S., Xu M., Marubayashi S., Zibinsky M., Hu D.X., Robles O., Jackson J.J., Baloche V., Busson P., Wustrow D., Brockstedt D.G., Talay O., Kassner P.D, & Cutler G. (2022). EBV+ tumors exploit tumor cell-intrinsic and -extrinsic mechanisms to produce regulatory T cell-recruiting chemokines CCL17 and CCL22. PLoS Pathogens, 18(1), e1010200.

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Induced Regulatory T Cell Generation

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Spleen and lymph nodes were obtained from 6-week-old to 8-week-old B6 Foxp3^GFP or BALB/c Foxp3^GFP mice and a single cell suspension was prepared. Red blood cells were removed using a 1× ACK lysis buffer (Gibco). CD4 cells were isolated by depleting CD25⁺ cells using the CD25 MicroBead Kit (Miltenyi Biotec) and subsequent enrichment of CD4 T cells using CD4 T Cell Isolation Kit (Miltenyi Biotech). Cells were cultured in complete Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific) with 1% non-essential amino acids (NEAA), 1% Penicillin-Streptomycin, 100 IU/mL L-glutamine and 10% Fetal Bovine Serum (FBS) (Gibco). The complete media was supplemented with 1× β-Mercaptoethanol (Thermo Fisher Scientific), 5 ng/mL of TGF-β (240-B-010, R&D Systems), 20 ng/mL of IL-2 (402 ML-020, R&D Systems), 10 µg/mL of anti-IFN-γ (BE0055, InvivoGen), 10 µg/mL of anti-IL-4 (BE0045, InvivoGen) and 1 µg/mL anti-CD28 (16-0281-86, Thermo Fisher Scientific) and cultured in 5 µg/mL anti-CD3 (16-0031-85, Thermo Fisher Scientific) coated plates at a concentration of 1×10⁶ cells/mL. On day 3, cells were cultured in RPMI medium with 20 ng/mL IL-2. Cells were harvested on day 7 for studies. Over 90% of iT_reg expressed CD25 and GFP.

Marshall L.A., Marubayashi S., Jorapur A., Jacobson S., Zibinsky M., Robles O., Hu D.X., Jackson J.J., Pookot D., Sanchez J., Brovarney M., Wadsworth A., Chian D., Wustrow D., Kassner P.D., Cutler G., Wong B., Brockstedt D.G, & Talay O. (2020). Tumors establish resistance to immunotherapy by regulating Treg recruitment via CCR4. Journal for Immunotherapy of Cancer, 8(2), e000764.

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Treg-mediated Suppression of T Effector Cells

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Bone marrow-derived dendritic cells (BMDC) were used as antigen presenting cells. Therefore, BM cells were prepared from femurs and tibiae of Thy1.2⁺ WT mice, and cultured for 7–8 days in complete RPMI medium supplemented with 5% culture supernatant of a granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing cell line [61] (link). Naïve CD25⁻CD4⁺ T effector cells were enriched from spleen and lymph nodes of Thy1.1⁺ mice by negative magnetic sorting using the Dynal Mouse CD4 negative T cell isolation kit (Invitrogen) and the CD25 MicroBead Kit (Miltenyi Biotec) following the manufacturer's protocol. FoxP3⁺CD4⁺ Tregs were isolated by FACS sorting of eGFP⁺CD4⁺ cells obtained from spleen and lymph nodes of M. bovis BCG infected or naive DEREG mice. Cultures were established in 96-well round bottom plates (Cellstar) in 200 µL complete RPMI medium using APC and different ratios of Treg:Teffector cells. Cultures were stimulated using 1 µg/mL of soluble αCD3 monoclonal antibody (clone 145-2C11, BioXCell). After 5 days, proliferation of CFSE (Life Technologies) -labelled T effector cells was determined by flow cytometry.

Berod L., Stüve P., Varela F., Behrends J., Swallow M., Kruse F., Krull F., Ghorbani P., Mayer C.T., Hölscher C, & Sparwasser T. (2014). Rapid Rebound of the Treg Compartment in DEREG Mice Limits the Impact of Treg Depletion on Mycobacterial Burden, but Prevents Autoimmunity. PLoS ONE, 9(7), e102804.

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T Cell Polarization and Checkpoint Regulation

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Naïve CD4⁺CD25^negCD62L⁺ T cells were isolated using CD4+ T cell isolation kit II (negative selection), CD25 microbead kit (negative selection), and CD62L microbeads (all from Miltenyi Biotec, Auburn, CA). Isolated CD4⁺CD25^negCD62L⁺ T cells were plated in flasks coated with αCD3 (5 μg/ml) in the presence of αCD28 (5 μg/ml) and Th1-(10 ng/ml IL-12, 1 μg/ml αIL-4), Th2- (10 ng/ml IL-4, μg/ml αIFNγ), or Th17- (20 ng/ml IL-6, 5 ng/ml TGFβ1, 10 ng/ml IL-23, 1 μg/ml αIL-4, 1 μg/ml αIFNγ) skewing cytokines. Cells were cultured for 5 days. Cultures were skewed or stimulated in the presence of either plate bound IgG-Fc (5 μg/ml), or plate bound PD-L1-Ig (R&D Systems, Minneapolis, MN). Cells were rested overnight in the absence of stimulation, and then restimulated on plates coated with various concentrations of αCD3, IgG-Fc and PD-L1-Ig. Cytokine production was assessed by ELISA using eBioscience mAbs.

McAlees J.W., Lajoie S., Dienger K., Sproles A.A., Richgels P.K., Yang Y., Khodoun M., Azuma M., Yagita H., Fulkerson P.C., Wills-Karp M, & Lewkowich I.P. (2015). Differential control of CD4+ T cell subsets by the PD-1/PD-L1 axis in allergic asthma. European journal of immunology, 45(4), 1019-1029.

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Isolation of Naive T-Cells and T-Cell Depleted Bone Marrow

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Total T-cells were isolated from naïve CB6F1 spleens by negative selection using mouse Pan T-Cell Isolation Kit II (Miltenyi Biotec, Auburn, CA) with a purity of >97%. CD25⁺ T-cells were depleted from this population using CD25 Microbead Kit (Miltenyi Biotec) resulting in preparations containing less than 0.4% remaining CD25⁺FoxP3⁺ cells. T-cells were depleted from BM cells using the CD3ε MicroBead Kit (Miltenyi Biotec), with less than 0.3% CD3ε⁺ cells remaining.

Stokes J., Hoffman E.A., Zeng Y., Larmonier N, & Katsanis E. (2016). Post-transplant bendamustine reduces GvHD while preserving GvL in experimental haploidentical bone marrow transplantation. British journal of haematology, 174(1), 102-116.

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Isolation and Labeling of Naive T Cells

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Isolation of Naive and Regulatory T Cells

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Single cell suspension of spleen and LN were collected, and naïve CD4⁺CD25⁻CD44^loCD62^hi T cells were isolated using a naïve CD4⁺ T cell isolation kit (Miltenyi Biotec). For the Foxp3 (GFP) knock-in mice, CD4⁺CD25⁻Foxp3 (GFP^-) T cells and CD4⁺CD25⁺Foxp3 (GFP⁺) nTreg were sorted by FACSAria (BD Biosciences) after purification using a CD4⁺ T cell isolation kit. In the indicated experiments, the CD4⁺ T cells were first enriched using a CD4⁺ T cell isolation kit, and CD25⁺ T cells were subsequently depleted using a CD25 microbead kit (Miltenyi Biotec) to obtain CD4⁺CD25⁻ T cells. The purity of cells sorted using this method was typically more than 95%.

Wang Q., He J., Flies D.B., Luo L, & Chen L. (2017). Programmed death one homolog maintains the pool size of regulatory T cells by promoting their differentiation and stability. Scientific Reports, 7, 6086.

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