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Cd25 m a251

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The CD25 (M-A251) is a monoclonal antibody that recognizes the human CD25 antigen, also known as the interleukin-2 receptor alpha chain (IL-2Rα). The CD25 antigen is expressed on activated T cells, B cells, and natural killer cells. This product is intended for research use only and its core function is to detect and quantify CD25-expressing cells.

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Lab products found in correlation

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7 protocols using cd25 m a251

1

Multiparametric Flow Cytometry Analysis

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PBMCs, either isolated from the peripheral blood or harvested after a 5-day MLC, were analyzed via cell-surface staining using CD3 (SP34), CD4 (L200), CD8 (SK1), CD20 (2H7), CD25 (M-A251) (all BD Pharmingen), CD16 (NKP15, BD Biosciences), and NKG2a (Z199, Beckman Coulter) antibodies. For chimerism analyses, we used an anti–MHC class I HLA mAb (H38, One Lambda, Inc.) that reacts specifically with an MHC class I antigen on donor but not recipient cells. To assess intracellular protein expression of FOXP3, cells were permeabilized using Fixation/Permeabilization solution (eBioscience) and then stained with anti-FOXP3 mAb (PCH101, BD Pharmingen). Cells were analyzed on a FACSverse (BD Biosciences) or Accuri Flow Cytometer (BD Biosciences) using FlowJo software (FLOWJO LLC).
Hotta K., Aoyama A., Oura T., Yamada Y., Tonsho M., Huh K.H., Kawai K., Schoenfeld D., Allan J.S., Madsen J.C., Benichou G., Smith R.N., Colvin R.B., Sachs D.H., Cosimi A.B, & Kawai T. (2016). Induced regulatory T cells in allograft tolerance via transient mixed chimerism. JCI Insight, 1(10), e86419.
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2

Analyzing Apoptosis in Treg Cells

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Human-specific antibodies used for flow cytometry included: CD4(RPA-T4), CD8((RPA-T8), CD25(M-A251), CD45RA(HI100), Annexin V(PE), 7-AAD(FITC) were purchased from BD Pharmingen, while FoxP3 (clone 249D) is from BioLegend and Ki67 is from eBioscience. The annexin V (PE)/7-AAD(FITC) were applied to assess the apoptosis of tTreg. Acquisition was performed using a CATON (BD Bioscience) and data were analyzed using FlowJo software (TreeStar).
Lu Y., Gao J., Zhang S., Gu J., Lu H., Xia Y., Zhu Q., Qian X., Zhang F., Zhang C., shen H., Hippen K.L., Blazar B.R., Lu L, & Wang X. (2018). miR-142-3p regulates autophagy by targeting ATG16L1 in thymic-derived regulatory T cell (tTreg). Cell Death & Disease, 9(3), 290.
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3

Activation and Phenotyping of T Cells

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Patients’ and healthy control (HC) PBMCs were purified by Ficoll density gradient centrifugation, washed with RPMI 1640 plus penicillin, streptomycin, and L-glutamine along with 10% FBS (R10) and filtered through a 40 µM strainer. Cells were resuspended to a concentration of 1 × 106 cells/mL and aliquoted (1 mL/well) into 48-well plates. PMA (final concentration 20 ng/mL), ionomycin (1 µM) and brefeldin A (5 µg/mL, Sigma-Aldrich) were added prior to incubation at 37°C for 5–6 hours. After incubation, cells were washed with FACS buffer and stained with LIVE/DEAD Blue (Life Technologies). Intracellular staining was performed using BD CytoFix/CytoPerm (BD Biosciences) reagents according to the manufacturer’s instructions using the following antibodies: CD3 (SK7, BD Biosciences), CD4 (RPA-T4, BD Biosciences), CD8 (SK1, BD Biosciences), CD45RO (UCHL1, Beckman Coulter), CXCR5 (RF8B2, BD Biosciences), CD25 (M-A251, BD Biosciences), IL-2 (MQ1–17H12, BioLegend), IL-4 (8D4–8, BD Biosciences), IL-5 (JES1–39D10, BD Biosciences), IL-13 (JES10-5A2, BD Biosciences), IFNγ (B27, BD Biosciences), IL-17A (eBio64DEC17, eBioscience). Cells were gated on viable CD3+ CD4+ CD45RO+ cells. Ex-vivo Treg staining was performed using anti-CD3 (UCHT1), CD4 (OKT4), CD25 (M-A251),, CD45RO (UCHL1), CD127 (HIL-7R–M21),, GATA3 (L50–823) and FOXP3 (236A/E7) antibodies as described 33 (link).
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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4

CARMIL2-expressing T cell analysis

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We identified the T cell populations in which CARMIL2 reexpression occurred by staining PBMCs with the Abs against the following: CD3 (SK7), CD4 (SK3), CD8 (RPA-T8), CD25 (M-A251, all from BD), CD27 (O323; eBioscience), CD45RA (HI100, BD), CD56 (HCD56; Biolegend), FoxP3 (PCH101; eBioscience), and CARMIL2 (EM53; Exbio). Viability was assessed with Zombie Aqua Live/Dead stain (Biolegend). In patients with CARMIL2-expressing T cell populations, T lymphoblasts were generated by stimulating 106 PBMCs with 5 ng/ml PMA, 1 µM ionomycin, and 100 U/ml IL-2 for 2 d before further expansion for 8–16 d in complete RPMI 1640 supplemented with 100 U/ml IL-2. The T lymphoblasts were sorted with Abs against CD3 (SK7), CD4 (SK3), CD8 (RPA-T8, all from BD), and CARMIL2 (EM53; Exbio). DNA was extracted from the CARMIL2-expressing cell populations with the DNeasy Blood and Tissue kit (Qiagen), and reversion events were confirmed by Sanger sequencing.
Lévy R., Gothe F., Momenilandi M., Magg T., Materna M., Peters P., Raedler J., Philippot Q., Rack-Hoch A.L., Langlais D., Bourgey M., Lanz A.L., Ogishi M., Rosain J., Martin E., Latour S., Vladikine N., Distefano M., Khan T., Rapaport F., Schulz M.S., Holzer U., Fasth A., Sogkas G., Speckmann C., Troilo A., Bigley V., Roppelt A., Dinur-Schejter Y., Toker O., Bronken Martinsen K.H., Sherkat R., Somekh I., Somech R., Shouval D.S., Kühl J.S., Ip W., McDermott E.M., Cliffe L., Ozen A., Baris S., Rangarajan H.G., Jouanguy E., Puel A., Bustamante J., Alyanakian M.A., Fusaro M., Wang Y., Kong X.F., Cobat A., Boutboul D., Castelle M., Aguilar C., Hermine O., Cheminant M., Suarez F., Yildiran A., Bousfiha A., Al-Mousa H., Alsohime F., Cagdas D., Abraham R.S., Knutsen A.P., Fevang B., Bhattad S., Kiykim A., Erman B., Arikoglu T., Unal E., Kumar A., Geier C.B., Baumann U., Neven B., Rohlfs M., Walz C., Abel L., Malissen B., Marr N., Klein C., Casanova J.L., Hauck F, & Béziat V. (2022). Human CARMIL2 deficiency underlies a broader immunological and clinical phenotype than CD28 deficiency. The Journal of Experimental Medicine, 220(2), e20220275.
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5

Immunophenotyping and Cytokine Analysis

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Peripheral blood (100 μL) was incubated with antibodies for 30 min at room temperature in the dark and then incubated with red blood cell lysis buffer and washed with phosphate-buffered saline (PBS). The cells were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, USA) according to the manufacturer’s instructions. The following antibodies (clones) were used for staining: CD3 (SP34-2), CD4 (L200), CD20 (2H7), CD8 (RPA-T8), and CD25 (M-A251), all from BD Bioscience (USA). All antibodies were titered in advance and used at optimal concentrations for flow cytometry. FlowJo v.10 was used to analyze the data. Measurement of cytokines and chemokines in the serum was performed using a MILLIPLEX® MAP NHP cytokine magnetic bead panel kit (Millipore Corporation, USA) and detected by a Bio-Plex 200 System (Bio-Rad Laboratories, USA).
Li H.Z., Li N., Wang J.J., Li H., Huang X., Guo L., Zheng H.W., He Z.L., Zhao Y., Yang Z.N., Fan H.T., Chu M.M., Yang J.X., Wu Q.W, & Liu L.D. (2020). Dysbiosis of gut microbiome affecting small intestine morphology and immune balance: a rhesus macaque model. Zoological Research, 41(1), 20-31.
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6

Erythrocyte Lysis and Leukocyte Staining

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Ammonium chloride was used to lyse erythrocytes from whole blood, as described previously [22 (link)]. Leukocytes were washed and stained using five different cocktails for flow cytometry. The following fluorochrome-labeled monoclonal antibodies were used: CD45 (2D1), CD16 (3G8), CD14 (M5E2), CD11c (B-ly6), CD56 (NCAM16.2), CD25 (M-A251), CD3 (OKT-3), CD4 (RPA-T4), CD8 (RPA-T8), TCRgamma/delta (g/d) (B1), CD45RA (HI100), CD62L (DREG-56), CD19 (HIB19), CD20 (2H7), CD27 (M-T271), lineage (LIN: CD3, CD14, CD16, CD19, CD20, CD56), HLA-DR (L243), CD123 (7G3) (all from BD Biosciences San Jose, CA), CD66b (G10F5), CD163 (GHI/61), TCR Va24-Ja18 (6B11), CD43 (10G7), CD10 (HI10a) (all from BioLegend Inc. San Diego, CA). Each staining well contained 3 million cells, and staining was done on ice for 30 minutes, followed by washing in PBS. Cells were fixed in 2% paraformaldehyde and analyzed flow cytometrically (FACSCanto II or FACSAria III, BD Biosciences, San Jose, CA), using FlowJo (TreeStar, Ashland, OR).
Prabhu S.B., Rathore D.K., Nair D., Chaudhary A., Raza S., Kanodia P., Sopory S., George A., Rath S., Bal V., Tripathi R., Ramji S., Batra A., Aggarwal K.C., Chellani H.K., Arya S., Agarwal N., Mehta U., Natchu U.C., Wadhwa N, & Bhatnagar S. (2016). Comparison of Human Neonatal and Adult Blood Leukocyte Subset Composition Phenotypes. PLoS ONE, 11(9), e0162242.
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7

Activation and Phenotyping of T Cells

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Patients’ and healthy control (HC) PBMCs were purified by Ficoll density gradient centrifugation, washed with RPMI 1640 plus penicillin, streptomycin, and L-glutamine along with 10% FBS (R10) and filtered through a 40 µM strainer. Cells were resuspended to a concentration of 1 × 106 cells/mL and aliquoted (1 mL/well) into 48-well plates. PMA (final concentration 20 ng/mL), ionomycin (1 µM) and brefeldin A (5 µg/mL, Sigma-Aldrich) were added prior to incubation at 37°C for 5–6 hours. After incubation, cells were washed with FACS buffer and stained with LIVE/DEAD Blue (Life Technologies). Intracellular staining was performed using BD CytoFix/CytoPerm (BD Biosciences) reagents according to the manufacturer’s instructions using the following antibodies: CD3 (SK7, BD Biosciences), CD4 (RPA-T4, BD Biosciences), CD8 (SK1, BD Biosciences), CD45RO (UCHL1, Beckman Coulter), CXCR5 (RF8B2, BD Biosciences), CD25 (M-A251, BD Biosciences), IL-2 (MQ1–17H12, BioLegend), IL-4 (8D4–8, BD Biosciences), IL-5 (JES1–39D10, BD Biosciences), IL-13 (JES10-5A2, BD Biosciences), IFNγ (B27, BD Biosciences), IL-17A (eBio64DEC17, eBioscience). Cells were gated on viable CD3+ CD4+ CD45RO+ cells. Ex-vivo Treg staining was performed using anti-CD3 (UCHT1), CD4 (OKT4), CD25 (M-A251),, CD45RO (UCHL1), CD127 (HIL-7R–M21),, GATA3 (L50–823) and FOXP3 (236A/E7) antibodies as described 33 (link).
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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