Treg suppression inspector human

Manufactured by Miltenyi Biotec

The Treg suppression inspector human is a flow cytometry-based assay that enables the identification and characterization of human regulatory T cells (Tregs) and their suppressive function. The core function of this product is to provide a standardized and reproducible method for the assessment of Treg-mediated immune suppression.

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Lab products found in correlation

Easysep human cd4 t cell enrichment kit, by STEMCELL (3 mentions) Celltrace violet, by Thermo Fisher Scientific (3 mentions) Magnetic bead, by Miltenyi Biotec (3 mentions) Anti cd25 beads, by Miltenyi Biotec (3 mentions) Celltrace cfse cell proliferation kit, by Thermo Fisher Scientific (2 mentions) Anti cd3, by Miltenyi Biotec (2 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions) Anti human cd25 microbeads, by Miltenyi Biotec (1 mentions) Live dead kit, by Thermo Fisher Scientific (1 mentions) Mojosort human cd4 t cell isolation kit, by BioLegend (1 mentions) Anti cd28, by Miltenyi Biotec (1 mentions) 7 aad, by BD (1 mentions) Facsaria, by BD (1 mentions) Automacs, by Miltenyi Biotec (1 mentions) Cd4 t cell isolation kit 2, by Miltenyi Biotec (1 mentions)

Spelling variants of this product

Treg Suppression Inspector (11 citations) Treg Suppression Inspector Human kit (2 citations) Human Treg Suppression Inspector beads (2 citations)

9 protocols using treg suppression inspector human

Treg-mediated Suppression of Tresponsive Cells

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CD4⁺ T cells were enriched using the EasySep Human CD4⁺ T cell enrichment kit (STEMCELL Technologies). CD4⁺CD25^hiCD127^lo Tregs and CD4⁺CD25⁻CD127⁺ responder T cells (Tresp) were sorted by fluorescence activated cell sorting (FACS) and then labeled with 5 μM of CFSE using the CellTrace CFSE Cell Proliferation kit (Invitrogen). Treg and Tresp cells were co-cultured at a 1:1 ratio in the presence of beads loaded with anti-CD2, anti-CD3, and anti-CD28 (Human Treg Suppression Inspector; Miltenyi Biotec). On day 4.5, proliferation of the Tresp cells was analyzed by flow cytometry measuring CFSE dilution.

Minto H., Mensah K.A., Reynolds P.R., Meffre E., Rubtsova K, & Gelfand E.W. (2019). A Novel ATM Mutation Associated with Elevated Atypical Lymphocyte Populations, Hyper-IgM, and Cutaneous Granulomas. Clinical immunology (Orlando, Fla.), 200, 55-63.

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Tresp and Treg Co-culture Assay

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Patient Tresp and corresponding patient Tregs were isolated and cultured in 96-well, round-bottom plates at a density of 5 × 10⁴ cells per well (in triplicate) at ratios of 1:1, 1:1/2, and 1:1/4 (Tresp:Tregs). A CD3/CD28 stimualtion reagent, Human Treg Suppression Inspector (Miltenyi Biotec), was added to the co-culture for five days followed by the addition of tritiated thymidine (Amersham Life Sciences) for 18 h. Proliferation is measured via tritium incorporation.

Thome A.D., Atassi F., Wang J., Faridar A., Zhao W., Thonhoff J.R., Beers D.R., Lai E.C, & Appel S.H. (2021). Ex vivo expansion of dysfunctional regulatory T lymphocytes restores suppressive function in Parkinson’s disease. NPJ Parkinson's Disease, 7, 41.

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Isolation and Functional Assay of Regulatory T Cells

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CD4+ T cells were isolated by negative selection using magnetic beads (Miltenyi Biotec). For isolation of CD4+CD25− effector T (Teff) cells, CD25+ cells were depleted using anti-CD25 beads (Miltenyi Biotec). Treg cells were isolated by sorting for CD4+CD25+CD127− cells. Teff cells were labeled with CellTrace Violet or CFSE (Life Technologies) and stimulated with anti-CD3, CD28, and CD2 coated beads (Treg suppression inspector human, Miltenyi Biotec), and autologous or allogeneic Treg cells were added at given ratios; cell divisions were evaluated by flow cytometry.

Ma C.A., Stinson J.R., Zhang Y., Abbott J.K., Weinreich M.A., Hauk P.J., Reynolds P.R., Lyons J.J., Nelson C.G., Ruffo E., Dorjbal B., Glauzy S., Yamakawa N., Arjunaraja S., Voss K., Stoddard J., Niemela J., Zhang Y., Rosenzweig S.D., McElwee J.J., DiMaggio T., Matthews H.F., Jones N., Stone K.D., Palma A., Oleastro M., Prieto E., Bernasconi A.R., Dubra G., Danielian S., Zaiat J., Marti M.A., Kim B., Cooper M.A., Romberg N.D., Meffre E., Gelfand E.W., Snow A.L, & Milner J.D. (2017). Germline hypomorphic CARD11 mutations in severe atopic disease. Nature genetics, 49(8), 1192-1201.

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Treg Suppression of Responder T Cells

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CD4⁺ T cells were enriched using the EasySep® Human CD4⁺T cell enrichment kit (STEMCELL). CD4⁺CD25^hiCD127^lo/- Tregs were sorted by flow cytometry whereas CD3⁺CD4⁺CD25^- Tresp cells were obtained after the depletion of CD25⁺ cells with anti-human CD25 microbeads (Miltenyi) and then labeled with CFSE at 5 μM. Treg and Tresp cells were co-cultured at a 1:1 ratio in the presence of beads loaded with anti-CD2, anti-CD3 and anti-CD28 (Treg suppression inspector human, Miltenyi). At 3.5-4.5 days, co-cultures were stained for viability with the LIVE/DEAD kit and proliferation of the viable Tresp was analyzed by CFSE dilution.

Romberg N., Virdee M., Chamberlain N., Oe T., Schickel J.N., Perkins T., Cantaert T., Rachid R., Rosengren S., Palazzo R., Geha R., Cunningham-Rundles C, & Meffre E. (2015). TNFRSF13B hemizygosity reveals TACI haploinsufficiency at later stages of B-cell development. The Journal of allergy and clinical immunology, 136(5), 1315-1325.

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Isolation and Functional Assay of Regulatory T Cells

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Assessing Treg Suppression of Teff Cells

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CD4⁺ T cells were isolated by negative selection using magnetic beads (Miltenyi Biotec). For isolation of CD4⁺CD25⁻ effector T (Teff) cells, CD25⁺ cells were depleted using anti-CD25 beads (Miltenyi Biotec). Treg cells were isolated by sorting for CD4⁺CD25⁺CD127⁻ cells. Teff cells were labeled with CellTrace Violet or CFSE (Life Technologies) and stimulated with anti-CD3, CD28, and CD2 coated beads (Treg suppression inspector human, Miltenyi Biotec), and autologous or allogeneic Treg cells were added at a ratio of 1:1; cell divisions were evaluated by flow cytometry.

Janssen E., Morbach H., Ullas S., Bannock J.M., Massad C., Menard L., Barlan I., Lefranc G., Su H., Dasouki M., Al-Herz W., Keles S., Chatila T., Geha R.S, & Meffre E. (2014). DOCK8 deficient patients have a breakdown in peripheral B cell tolerance and defective regulatory T cells. The Journal of allergy and clinical immunology, 134(6), 1365-1374.

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Treg-Mediated Suppression of Effector T Cells

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CD4⁺ T cells were enriched using the EasySep® Human CD4⁺ T cell enrichment kit (STEMCELL) or MojoSort™ Human CD4 T Cell Isolation Kit (Biolegend). CD4⁺CD25^hiCD127^lo/- Tregs and CD3⁺CD4⁺CD25^- Tresp cells were sorted by flow cytometry and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Thermo Fischer). Treg and Tresp cells were co-cultured at a 1:1 ratio with beads loaded with anti-CD2, anti-CD3 and anti-CD28 (Treg suppression inspector human, Miltenyi). Co-cultures were stained for viability with the LIVE/DEAD kit (Thermo Fisher), and proliferation of viable Tresp cells analyzed for CFSE dilution at 3.5-4.5 days.

Romberg N., Le Coz C., Glauzy S., Schickel J.N., Trofa M., Nolan B.E., Paessler M., Xu M.L., Lambert M., Lakhani S.A., Khokha M.K., Jyonouchi S., Heimall J., Takach P., Maglione P.J., Catanzaro J., Hsu F.I., Sullivan K.E., Cunningham-Rundles C, & Meffre E. (2018). CVID patients with autoimmune cytopenias exhibit hyperplastic yet inefficient germinal center responses. The Journal of allergy and clinical immunology, 143(1), 258-265.

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CFSE-Based T Cell Proliferation Assay

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Peripheral blood mononuclear cells (PBMCs) obtained from healthy donors were labeled with 1 µmol/L carboxyfluorescein succinimidyl ester (CFSE; Thermo Fisher Scientific) according to the manufacturer's protocol. CFSE‐labeled PBMCs (1 × 10⁵) were plated into 96‐well plates with transduced HSC‐3 cells (2 × 10⁴) in RPMI medium supplemented with 10% FCS, 100 units/mL penicillin, and 100 μg/mL streptomycin. Following the addition of anti‐CD3/anti‐CD28 stimulus (Treg Suppression Inspector human; Miltenyi Biotec), the cells were incubated for 96 h. After incubation, floating cells were harvested and stained with allophycocyanin (APC)‐CD3 antibody (BD Bioscience) at 4°C for 1 h. Then, 7‐AAD (BD Bioscience) was added 10 min before the analysis. The proliferation of CD3‐positive 7‐AAD‐negative cells was measured by dilution of CFSE staining using flow cytometry.

Takahashi H., Rokudai S., Kawabata‐Iwakawa R., Sakakura K., Oyama T., Nishiyama M, & Chikamatsu K. (2021). AKT3 is a key regulator of head and neck squamous cell carcinoma. Cancer Science, 112(6), 2325-2334.

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Isolation and Proliferation Analysis of Regulatory T Cells

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Peripheral blood samples for the suppression and proliferation analysis were sourced from controls and KTR (stable >1 year) who required venesection but were otherwise well: the KTR for polycythaemia; the controls for iron overload. PBMC were obtained by Ficoll gradient and CD4 + T cells purified using the CD4 + T cell Isolation Kit II and AUTOMACS (Miltenyi Biotec Australia, Macquarie Park, NSW, Australia). CD4 + cells were stained as described above and CD4 + CD25 + CD39 + mTreg and CD4 + CD25 -CD39 -nTeff populations were isolated following flow cytometry cell sorting (FACS Aria, BD Biosciences, San Jose, CA). nTeff cells were stained with CFSE using the CellTrace™ CFSE Cell Proliferation Kit (Molecular Probes™, Thermo Scientific, Melbourne, Australia). nTeff were activated using Treg Suppression Inspector, human (Miltenyi Biotec). A total of 5 x 10 4 nTeff cells/well This article is protected by copyright. All rights reserved.

McRae J.L., Chia J.S., Pommey S.A, & Dwyer K.M. (2017). Evaluation of CD4⁺ CD25^+/- CD39⁺ T-cell populations in peripheral blood of patients following kidney transplantation and during acute allograft rejection. Nephrology (Carlton, Vic.), 22(7).

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