Cd4 cd25 cd127dim regulatory t cell isolation kit 2

Manufactured by Miltenyi Biotec

Sourced in Germany, United States

The CD4+CD25+CD127dim/- Regulatory T Cell Isolation Kit II is a laboratory product designed to isolate CD4+CD25+CD127dim/- regulatory T cells from human peripheral blood mononuclear cells (PBMCs) or other cell sources. The kit utilizes magnetic bead-based cell separation technology to enrich the target cell population.

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Close spelling variants of this product

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

14 protocols using cd4 cd25 cd127dim regulatory t cell isolation kit 2

Isolation and Culture of T-Cell Subsets

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For the isolation of CD4⁺ cells, the CD4⁺ T-Cell Isolation Kit human from Miltenyi Biotec was used according to the manufacturer’s instructions and the purity was analysed by flow cytometry. For the isolation of CD4⁺ CD25⁺ (Foxp3⁺) Treg cells, the CD4⁺ CD25⁺ CD127^dim/⁻ Regulatory T-Cell Isolation Kit II human from Miltenyi Biotec was used to separate Treg and Tconv from pre-enriched CD4⁺ T cells. The purity of the Treg was >95% (CD25⁺ Foxp3⁺) as analysed by flow cytometry. In brief, 3 × 10⁵ Treg or Tconv per condition were then cultured for 2 h at 37°C/5% CO₂ in the absence or presence of sertraline at 0.2, 1, 5 and 10 µM. After the short-term culture, the T cells were disrupted by repeated freezing and thawing and further processed as described earlier.⁷ (link)

Wiese T., Dennstädt F., Hollmann C., Stonawski S., Wurst C., Fink J., Gorte E., Mandasari P., Domschke K., Hommers L., Vanhove B., Schumacher F., Kleuser B., Seibel J., Rohr J., Buttmann M., Menke A., Schneider-Schaulies J, & Beyersdorf N. (2021). Inhibition of acid sphingomyelinase increases regulatory T cells in humans. Brain Communications, 3(2), fcab020.

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Isolation of Human CD4+CD25+CD127-/dim Treg Cells

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Sera were obtained after centrifugation of clotted blood samples and stored at −20°C until further analysis. PBMCs were isolated from heparinized samples using Ficoll-Hypaque (GE Healthcare Life Sciences) gradient separation. CD4⁺CD25⁺CD127^−/dim T cells were purified using the CD4⁺CD25⁺CD127^−/dim regulatory T Cell Isolation Kit II (Miltenyi Biotec, Auburn, CA, USA). The obtained cell populations had a purity of 95% as shown by flow cytometry on immunostained cells.

Cicco S., Desantis V., Vacca A., Cazzato G., Solimando A.G., Cirulli A., Noviello S., Susca C., Prete M., Brosolo G., Catena C., Lamanuzzi A., Saltarella I., Frassanito M.A., Cimmino A., Ingravallo G., Resta L., Ria R, & Montagnani M. (2022). Cardiovascular Risk in Patients With Takayasu Arteritis Directly Correlates With Diastolic Dysfunction and Inflammatory Cell Infiltration in the Vessel Wall: A Clinical, ex vivo and in vitro Analysis. Frontiers in Medicine, 9, 863150.

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Isolation and Purification of Regulatory T Cells

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Peripheral blood mononuclear cells (PBMCs) and cord blood mononuclear cells (CBMCs) were isolated by density gradient centrifugation with Ficoll-Hypaque (GE Healthcare, Little Chalfont, UK). Next, Treg cells were isolated from PBMCs and CBMCs using the CD4⁺ CD25⁺ CD127^dim/− regulatory T cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany), because identifying the expression of CD127 in combination with CD4 and CD25 is a reliable strategy to discriminate Treg cells (CD127^dim/−) from conventional T cells [24 (link),25 (link)], according to the manufacturer's instructions. CD4⁺ CD25⁺ CD127^dim/− cells were negatively purified by a cocktail of biotin-conjugated antibodies and anti-biotin monoclonal antibodies conjugated to MicroBeads. Then, CD4⁺ CD25⁺ CD127^dim/− Treg cells were directly labelled with CD25 MicroBeads and isolated using a selection column (Miltenyi Biotec). Simultaneously, we also separated CD25⁺ and CD25⁻ T cells from PBMCs according to those described by Yates et al. [26 (link)]. For CCR9⁺ CD4⁺ T and CCR9⁺ CD4⁺ CD25⁺ CD127^dim/− Treg cell isolation, PBMCs, CBMCs or CD4⁺ CD25⁺ CD127^dim/− Treg cells were stained with fluorochrome-conjugated anti-human antibodies against CD3, CD4, and CCR9 (Table S1) and sorted using the Aria II cell sorter (BD Biosciences, San Jose, CA, USA). The purity of all isolated cell subsets was >90%.

Ma F., Li S., Gao X., Zhou J., Zhu X., Wang D., Cai Y., Li F., Yang Q., Gu X., Ge W., Liu H., Xiao X, & Hao H. (2019). Interleukin-6-mediated CCR9+ interleukin-17-producing regulatory T cells polarization increases the severity of necrotizing enterocolitis. EBioMedicine, 44, 71-85.

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PBMC Isolation and T Cell Purification

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Peripheral blood mononuclear cells (PBMCs) were isolated from fresh, whole blood by density gradient centrifugation using Ficoll-Hypaque (Sigma-Aldrich, St Louis, MO, USA). CD4+ T cells and CD4+CD25+CD127dim/- Tregs were purified from PBMC using a CD4+ Cell Isolation Kit (Miltenyi, Bergisch Gladbach, Germany) and CD4+CD25+CD127dim/- Regulatory T Cell Isolation Kit II (Miltenyi) according to the manufacturer’s instruction, respectively.

Sanz-Rubio D., Sanz A., Varona L., Bolea R., Forner M., Gil A.V., Cubero P., Marin-Oto M., Martin-Burriel I, & Marin J.M. (2020). Forkhead Box P3 Methylation and Expression in Men with Obstructive Sleep Apnea. International Journal of Molecular Sciences, 21(6), 2233.

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Isolation and Purification of Regulatory T Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll-Hypaque (Sigma-Aldrich, St Louis, MO, USA). CD4⁺CD25⁺CD127^dim/− Tregs were purified using CD4⁺CD25⁺CD127^dim/− regulatory T cell isolation kit II (Miltenyi, Bergisch Gladbach, Germany). CD8⁺ T cells were purified using human CD8⁺ T cell isolation kit (Miltenyi). The purity of enrich cells was more than 90% by flow cytometry determination.

Shao X., Ma J., Jia S., Yang L., Wang W, & Jin Z. (2017). Interleukin-35 Suppresses Antiviral Immune Response in Chronic Hepatitis B Virus Infection. Frontiers in Cellular and Infection Microbiology, 7, 472.

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Evaluating Treg Cell Suppressive Function

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After 3 days of coculture with iPSC-MSCs, Treg cells were isolated from the cocultured PBMCs using the CD4⁺CD25⁺CD127^dim/− Regulatory T Cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). The isolated Treg cells were further cocultured at a ratio of 1:2 with 5 × 10⁵ allogeneic CFSE-stained PBMCs for another 3 days in the presence of PHA (5 μg/ml; Sigma) in a 24-well-plate, and then the stained PBMCs’ proliferation was determined by flow cytometry analysis, to examine the inhibitory function of the Treg cells. CD4⁺CD25⁺CD127^dim/− cells from PBMCs without coculture were as controls.

Fan X.L., Zeng Q.X., Li X., Li C.L., Xu Z.B., Deng X.Q., Shi J., Chen D., Zheng S.G, & Fu Q.L. (2018). Induced pluripotent stem cell-derived mesenchymal stem cells activate quiescent T cells and elevate regulatory T cell response via NF-κB in allergic rhinitis patients. Stem Cell Research & Therapy, 9, 170.

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Isolation of Murine and Human T-cell Subsets

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Murine T cells were FACS-sorted from wild-type (WT) or CD137^−/− C57BL/6 murine splenocytes according to surface markers: CD4⁺CD25⁻ (conventional T cells (Tcon)) or CD4⁺CD25⁺ (Treg). Human Tregs and CD8⁺ T cells were isolated from healthy donor PBMC by CD4⁺CD25⁺CD127^dim/− Regulatory T Cell Isolation Kit II and CD8⁺ T Cell Isolation Kit, respectively (Miltenyi Biotec, Bergisch Gladbach, Germany).

Luu K., Patwardhan M.V., Zeng Q., Wickström S.L., Lundqvist A, & Schwarz H. (2021). Regulatory T Cells Inhibit T Cell Activity by Downregulating CD137 Ligand via CD137 Trogocytosis. Cells, 10(2), 353.

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Treg-mediated Inhibition of Cytokine Secretion

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Human PBMCs were isolated from the peripheral blood of healthy donors by standard density gradient centrifugation on Ficoll-Hypaque (GE Healthcare-Life Sciences, USA). Regulatory T cells (Tregs) were isolated from PBMCs using magnetic beads technology (CD4⁺ CD25⁺ CD127^dim/- Regulatory T Cell Isolation Kit II; Miltenyi Biotec, USA) according to the manufacturer’s recommendations. Autologous PBMCs were stimulated with soluble anti-CD3 and anti-CD28 antibodies (1 and 0.5 µg/mL respectively; BD Biosciences, USA) in the presence of Tregs (1:1 PBMC to Treg ratio). The therapeutic antibodies were added, and after 3 days of culture, cytokine levels were measured in the culture supernatant using ProcartaPlex immunoassay according to the manufacturer’s recommendations (Th1/Th2/Th9/Th17/Th22/Treg Cytokine 18-Plex Human ProcartaPlex™ Panel; Thermo Fisher Scientific). Results were expressed as a percentage of inhibition of cytokine secretion relative to the level secreted in the absence of Tregs.

Geuijen C., Tacken P., Wang L.C., Klooster R., van Loo P.F., Zhou J., Mondal A., Liu Y.B., Kramer A., Condamine T., Volgina A., Hendriks L.J., van der Maaden H., Rovers E., Engels S., Fransen F., den Blanken-Smit R., Zondag-van der Zande V., Basmeleh A., Bartelink W., Kulkarni A., Marissen W., Huang C.Y., Hall L., Harvey S., Kim S., Martinez M., O’Brien S., Moon E., Albelda S., Kanellopoulou C., Stewart S., Nastri H., Bakker A.B., Scherle P., Logtenberg T., Hollis G., de Kruif J., Huber R., Mayes P.A, & Throsby M. (2021). A human CD137×PD-L1 bispecific antibody promotes anti-tumor immunity via context-dependent T cell costimulation and checkpoint blockade. Nature Communications, 12, 4445.

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Isolation of Human Regulatory T Cells

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Tregs were isolated from PBMCs using the CD4⁺CD25⁺CD127^dim/– Regulatory T Cell Isolation Kit II (cat.#130-094-775, Miltenyi Biotec) according to the manufacturer’s instructions. Briefly, non-CD4⁺ and CD127^high cells were indirectly magnetically labeled with a cocktail of biotin-conjugated antibodies, and anti-biotin monoclonal antibodies conjugated to MicroBeads. The labeled cells were subsequently depleted by separation through MACS^® column. In the second step, the CD4⁺CD25⁺CD127^dim/– Tregs were directly labeled with CD25 MicroBeads II and isolated by positive selection from the pre-enriched CD4⁺ T-cell fraction, by separation through MACS^® Column and eluted as the positively selected cell fraction.

Grigoriou M., Banos A., Hatzioannou A., Kloetgen A., Kouzis P., Aggouraki D., Zakopoulou R., Bamias G., Kassi E., Mavroudis D., Bamias A., Boumpas D.T., Tsirigos A., Gogas H., Alissafi T, & Verginis P. (2021). Regulatory T cell transcriptomic reprogramming characterizes adverse events by checkpoint inhibitors in solid tumors. Cancer immunology research, 9(7), 726-734.

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Isolation of CD4+ T Cells and Tregs

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EDTA anticoagulant peripheral bloods were collected from each patients. PBMCs were isolated by density gradient centrifugation using Ficoll-Hypaque (Sigma-Aldrich, St Louis, MO, USA). CD4⁺ T cells or CD4⁺CD25⁺CD127^dim/- Tregs were purified from PBMC using CD4⁺ Cell Isolation Kit (Miltenyi, Bergisch Gladbach, Germany) or CD4⁺CD25⁺CD127^dim/- Regulatory T Cell Isolation Kit II (Miltenyi) according to manufacturer’s instruction, respectively. The purity of enriched cells was more than 95% by flow cytometry determination.

Yang L., Jia S., Shao X., Liu S., Zhang Q., Song J., Wang W, & Jin Z. (2019). Interleukin-35 modulates the balance between viral specific CD4+CD25+CD127dim/- regulatory T cells and T helper 17 cells in chronic hepatitis B virus infection. Virology Journal, 16, 48.

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