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Cd25 pe cy7

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The CD25 PE-Cy7 is a flow cytometry reagent that detects the expression of the CD25 antigen on the surface of cells. CD25 is the alpha subunit of the interleukin-2 receptor, which is a marker for activated T cells and regulatory T cells. The PE-Cy7 fluorochrome is used to label the anti-CD25 antibody, enabling its detection and quantification on cells by flow cytometry.

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Lab products found in correlation

Cd4 percp efluor710, by Thermo Fisher Scientific (5 mentions) Cd44 apc efluor780, by Thermo Fisher Scientific (5 mentions) Cd62l efluor450, by Thermo Fisher Scientific (5 mentions) Cd25 pe, by Thermo Fisher Scientific (4 mentions) Cd45.1 fitc, by Thermo Fisher Scientific (4 mentions) Tcrβ apc, by Thermo Fisher Scientific (4 mentions) Cd45.2 fitc, by Thermo Fisher Scientific (4 mentions) Cd45.2 alexafluor700, by Thermo Fisher Scientific (4 mentions) Cd25 efluor450, by Thermo Fisher Scientific (4 mentions) Tcr β percp cy5.5 cd4 bv711, by BioLegend (4 mentions) Cd44 bv785, by BioLegend (4 mentions) Cd25 bv650, by BioLegend (4 mentions) Cd62l buv737, by BD (4 mentions) Ctla 4 apc, by Thermo Fisher Scientific (3 mentions) Cd4 percp cy5, by Thermo Fisher Scientific (3 mentions) Cd45.2 pe dazzle, by BioLegend (3 mentions) Nk1.1 pe cy7, by Thermo Fisher Scientific (3 mentions) Cd45.1 bv650, by Thermo Fisher Scientific (3 mentions) Anti mouse ki67 fitc or pe, by Thermo Fisher Scientific (3 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (3 mentions)

27 protocols using cd25 pe cy7

1

Multiparametric Flow Cytometry for Immune Profiling

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For analysis of PD-1, cells were washed in serum-free PBS and stained with a fixable viability dye, eFluor506, CD4-PerCPCy5.5, PD1-PECY7, CD8-APCeFluor780 (eBioscience), incubated for 15 min at room temperature in the dark, and then washed in PBS buffer containing 1% FBS and sodium azide. Cells were stained for Treg cell markers using a FoxP3 staining buffer set (eBioscience) and accompanying protocol. Treg cell markers included CD39-FITC, FoxP3-PE, CD73-PerCPeFluor710, CD25-PECY7, CTLA-4-APC, CD127-APCeFluor780, Ki67-eFluor450 (eBioscience), and CD4-V500 (BD Biosciences). An intracellular staining kit (Fix and Perm kit, Invitrogen) was used to analyze cytokine production after restimulation with PMA/ionomycin. Cells were stained with IL-17A-AlexaFluor488, IL-10-PE, TNF-α-PerCPCy5.5, CD45RA-PECY7, CD8-APCeFluor780, FoxP3-eFluor450 (all eBioSciences), CD45 AlexaFluor700 (BioLegend), IFN-γ-APC, CD3-V500, and IL-2-PE-CF594 (BD Biosciences). Due to PMA/ionomycin-mediated reduction in CD4 expression, CD4+ T cells were identified as CD3+CD8− T cells for cytokine analysis. Cells were acquired on a BD LSRFortessa flow cytometer and analyzed using FlowJo software (Flowjo LLC).
Dunne M.R., Ryan C., Nolan B., Tosetto M., Geraghty R., Winter D.C., O’Connell P.R., Hyland J.M., Doherty G.A., Sheahan K., Ryan E.J, & Fletcher J.M. (2016). Enrichment of Inflammatory IL-17 and TNF-α Secreting CD4+ T Cells within Colorectal Tumors despite the Presence of Elevated CD39+ T Regulatory Cells and Increased Expression of the Immune Checkpoint Molecule, PD-1. Frontiers in Oncology, 6, 50.
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2

CSF Immune Cell Profiling by Flow Cytometry

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Five milliliters of CSF were obtained by lumbar spinal tap and centrifuged at 550g at 4°C for 5 minutes. The supernatant was removed, and the pellet was resuspended and incubated at 4°C for 20 minutes with fluorochrome-labeled antibodies against CD4, CD8, CD20, CD25, C-C chemokine receptor type 6 (CCR6), HLA-DR, CD14, and CD45 (BD Pharmingen Mouse Anti-Human Antibodies: CD4 PerCP, CD8 FITC, CD 20 Alexa Fluor 700, CD25 PE-Cy7, CD196 (CCR6) PE, Anti-HLA-DR V450, and CD14 APC; eBioscience mouse Anti-Human Antibody: CD45 EF 605). Cells were washed and resuspended in 200 μL of fluorescence-activated cell sorting (FACS) buffer before FACS analysis on a FACSCanto-II. Analysis of cell subset counts was performed with FlowJo. All CSF and serum tests were performed with researchers blinded to the clinical diagnosis and MRI results. CSF samples were measured by flow cytometry in a time frame of 90 minutes after lumbar spinal tab. CSF supernatants were stored in polypropylene tubes at −80°C until batched analysis of NfL and CHI3L1 concentrations.
Engel S., Friedrich M., Muthuraman M., Steffen F., Poplawski A., Groppa S., Bittner S., Zipp F, & Luessi F. (2019). Intrathecal B-cell accumulation and axonal damage distinguish MRI-based benign from aggressive onset in MS. Neurology® Neuroimmunology & Neuroinflammation, 6(5), e595.
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3

Comprehensive Flow Cytometry of Immune Subsets

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Spleens were smashed and filtered through 40 μm gauze (BD Biosciences). Single-cell suspensions were then stained for flow cytometry with the following antibodies: CD4 Pacific blue (clone GK1.5), CD25 PE-cy7 (clone PC61), FoxP3 AlexaFluor (clone FJK-16a, eBioscience) were used in combination with a FoxP3 staining kit (eBioscience). TFH cells were detected with the following antibodies: CD4 (clone RM4-5), B220 (clone RA3-6B2), CXCR5 (clone SPRCL5), PD-1 (clone 29 F.1A12), ICOS (clone 7E.17G9), Bcl6 (clone K112-91), and IL-21 (clone FFA21). Further antibodies: CD3 (clone 145-2C11), CD8α (clone 53-6.7), CD11b (clone M1/70), CD11c (clone N418), CD45 (clone 30-F11), CD138 (clone 281-2), F4/80 (clone BM8), Fas (clone 15A7), Ly6c (clone HK1.4), Ly6G (clone 1A8), MHCII (clone M5/114.15.2), TACI (clone ebio8F10-3), and Siglec F (clone ES22-10D8), PNA-biotin (Vector), Streptavidin-PE/CF594 (BD), NKp46 (clone 29A1.4), CD49 (clone DX5), CD16 (clone 93), IL-4 (clone 11B11), IL-17 (clone TC11-18H10.1), IL-9 (clone RM9A4). TFH:B cell doublet gating strategy is adopted from previously published article69 (link) and is shown in Supplementary Fig. 3f.
Azizov V., Dietel K., Steffen F., Dürholz K., Meidenbauer J., Lucas S., Frech M., Omata Y., Tajik N., Knipfer L., Kolenbrander A., Seubert S., Lapuente D., Sokolova M.V., Hofmann J., Tenbusch M., Ramming A., Steffen U., Nimmerjahn F., Linker R., Wirtz S., Herrmann M., Temchura V., Sarter K., Schett G, & Zaiss M.M. (2020). Ethanol consumption inhibits TFH cell responses and the development of autoimmune arthritis. Nature Communications, 11, 1998.
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4

Multi-parameter Flow Cytometry of Lymphocyte Subsets

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Multi-parameter flow cytometry analysis of different cell subsets was performed using PBMC from patients at baseline and at cycle 3. Cells were stained with different antibody panels to study different lymphocyte populations. To study T regulatory (Treg) cells we used the following panel: CD3-AmCyan, Foxp3-efluor450 (eBioscience, CA), CD127-FITC, ICOS-PE (eBioscience, CA), CD4-PerCP-Cy5.5, CD39-APC, CD25-PE-Cy7 and CD8-APC-H7. For the proliferation panel we stained for CD3-AmCyan, Foxp3-efluor450, KI-67-Alexa Fluor 488, ICOS-PE, CD4-PerCP-Cy5.5, CD39-APC, CD25-PE-Cy7 and CD8-APC-H7. The frequencies of the different populations were translated into cell numbers (# cells/µl of blood) using Absolute Lymphocyte Counts (ALC). All antibodies are from BD Biosciences, unless otherwise indicated. The acquisition was carried out on a FACS Canto II flow cytometer (BD Biosciences, CA). All analysis was done with the software FlowJo (Tree Star, OR).
Plimack E.R., Desai J.R., Issa J.P., Jelinek J., Sharma P., Vence L.M., Bassett R.L., Ilagan J.L., Papadopoulos N.E, & Hwu W.J. (2014). A phase I study of decitabine with pegylated interferon α-2b in advanced melanoma: impact on DNA methylation and lymphocyte populations. Investigational new drugs, 32(5), 969-975.
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5

Multiparameter Immunophenotyping of Cells

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Cells were immunostained with the following mAbs: CD31-FITC, CD144-vioblue, KDR-PE-Vio770, CD4-FITC, APC and Vioblue, Foxp3-APC, CD62L-PE, ICOS–PE, CTLA4–biotin or PE, IFNγ–APC, TNFα-FITC or PE, IL-10-APC, IL-17-PE, IL-2-FITC, HLA-G-PE, TGFβ-biotin, REA-control-APC, PE, PE-Cy5 and PE-Cy7, CD8α-FITC or Percp or PE-Cy7 and TNFR2-APC and PE (Miltenyi) streptavidin-PE-Cy7 or PE-Cy5, Foxp3-PE-Cy5, CD25-PE-Cy7 (eBioscience) and TGFβ-PE (Biolegend). Intracellular Foxp3 staining was performed according to the manufacturer’s instructions, using Foxp3 staining buffer set from eBioscience. Events acquired on a LSRFORTESSA flow cytometer (BD-Biosciences) and analyzed using FlowJo software v10 (FlowJo-LLC).
Naserian S., Abdelgawad M.E., Afshar Bakshloo M., Ha G., Arouche N., Cohen J.L., Salomon B.L, & Uzan G. (2020). The TNF/TNFR2 signaling pathway is a key regulatory factor in endothelial progenitor cell immunosuppressive effect. Cell Communication and Signaling : CCS, 18, 94.
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6

Characterizing T Cell Subsets in Mice

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Single cell suspensions were prepared from the thymus, spleen and lymph nodes of busulfan chimeric mice, wildtype control mice, or germ free mice. Cells were stained with the following monoclonal antibodies and cell dyes: CD45.1 FITC, CD45.2 FITC, CD45.2 AlexaFluor700, TCR- β APC, CD4+ PerCP-eFluor710, CD44 APC-eFluor780, CD25 PE, CD25 eFluor450, CD25 PE-Cy7, CD62L eFluor450, NK1.1 PE-Cy7 (all eBioscience), CD45.1 BV650, CD45.2 PE-Dazzle, TCR- β PerCP-Cy5.5 CD4+ BV711, CD44 BV785, CD25 BV650 (all Biolegend), CD62L BUV737 (BD Biosciences), LIVE/DEAD nearIR and LIVE/DEAD blue viability dyes. For Ki67 staining, cells were fixed using the eBioscience Foxp3/Transcription Factor Staining Buffer Set and stained with either anti-mouse Ki67 FITC or PE (both eBioscience). Cells were acquired on a BD LSR-II or a BD LSR-Fortessa flow cytometer and analysed with Flowjo software (Treestar). Conventional CD4+ cells were identified as live TCR- β + CD4+ CD25- NK1.1-, and then CD44 and CD62L were used to identify EM (CD44+CD62L-) and CM (CD44+CD62L+) subsets.
Hogan T., Nowicka M., Cownden D., Pearson C.F., Yates A.J, & Seddon B. (2019). Differential impact of self and environmental antigens on the ontogeny and maintenance of CD4+ T cell memory. eLife, 8, e48901.
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7

Induction and Phenotyping of Tregs

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The same procedure was followed as described in DC‐T cell co‐culture procedure and maintained for up to 5 days for induction of T regulatory cells (Tregs). On the 5th day, cells were harvested and stained for Treg‐specific markers; anti‐mouse CD4‐FITC (# 11‐0042‐82), CD25‐PE‐Cy7 (# 25‐0251‐82), and FOXP3‐PE (# 12–5773‐82) all from eBioscience (San Diego, CA); and CD127‐APC (# 158205) Biolegend (San Diego, CA) fluorochrome‐tagged monoclonal antibodies.
Bashir H., Singh S., Singh R.P., Agrewala J.N, & Kumar R. (2023). Age‐mediated gut microbiota dysbiosis promotes the loss of dendritic cells tolerance. Aging Cell, 22(6), e13838.
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8

T-cell Activation Assay Protocol

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For T-cell activation assays, lymphocytes in mixed splenocyte suspensions were counted using a haemocytometer and 2 × 105 cells plated per 96 well, in complete RPMI media (RPMI with 10% heat inactivated (HI) foetal bovine serum (FBS), 100 U/mL penicillin and streptomycin, and 5 µM 2-mercaptoethanol (all Invitrogen)) supplemented with or without soluble 2 µg/mL anti-CD3e (clone 145-2C11) and 2 µg/mL anti-CD28 (clone 37.51), to activate T cells. Cells were incubated for 24, 48, and 72 h in a humidified 37 °C, 5% CO2 incubator. To assay for activation markers, cells were analysed by flow cytometry with antibodies to CD4-PE, CD8-APC, CD25-PECy7, and CD69-FITC (clone H1.2F3, eBiosciences). See Supplementary Fig. 1d and e for representative FACS plots. For cytokine analysis, CD4+ T cell from spleen were FACS sorted and plated as above. Cells were incubated for 24 and 48 h, and then supernatants collected for cytokine analysis using ELISA assays for IL-2, IFN-γ, as per the manufacturer’s instructions (eBioscience).
Patel S.P., Randle S.J., Gibbs S., Cooke A, & Laman H. (2016). Opposing effects on the cell cycle of T lymphocytes by Fbxo7 via Cdk6 and p27. Cellular and Molecular Life Sciences, 74(8), 1553-1566.
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9

Comprehensive Immune Cell Profiling

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An LSR II (BD Biosciences) was used for flow cytometry. The following antibodies were used: LIVE/DEAD Aqua (Life Technologies, L34957), CD3 APC/Cy7 (BioLegend, 300317), CD3e PerCP/Cy5.5 (BD, 561108), CD4 PB (Invitrogen, MHCD0428), CD8 BV605 (BD, 564115), CD25 PE-Cy7 (eBioscience, 25025942), Foxp3 PE (eBioscience, 12477182), IFN-γ PE-Cy7 (eBioscience, 25731141), TNF-α FITC (BD, 554418), IL-2 APC (BD, 562041), F4/80 PE-Cy7 (BioLegend, 123113), CD16/CD32 APC (eBioscience, 17016181), CD16-2/FCGR4 PE (SinoBiological, 50036R012P10), CD11b FITC (eBiosciences, 11011281), CD45 AF700 (BioLegend, 103127).
Patel M.A., Kim J.E., Theodros D., Tam A., Velarde E., Kochel C.M., Francica B., Nirschl T.R., Ghasemzadeh A., Mathios D., Harris-Bookman S., Jackson C.C., Jackson C., Ye X., Tran P.T., Tyler B., Coric V., Selby M., Brem H., Drake C.G., Pardoll D.M, & Lim M. (2016). Agonist anti-GITR monoclonal antibody and stereotactic radiation induce immune-mediated survival advantage in murine intracranial glioma. Journal for Immunotherapy of Cancer, 4, 28.
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10

Multiparameter Flow Cytometry Panel

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The following monoclonal antibodies and cell dyes were used: CD45.1 FITC, CD45.2 AlexaFluor700, CD45.2 FITC, TCR-beta APC, CD4 PerCP-eFluor710, CD44 APC-eFluor780, CD25 PE-Cy7, L-selectin eFluor450, CD122 biotin (all eBioscience); CD8 Pacific Orange, streptavidin PE-TexasRed, LIVE/DEAD blue (all Invitrogen); and CD45.1 Brilliant Violet 650, CD4 Brilliant Violet 711, and TCR-beta PerCP-Cy5.5 (all BioLegend). Samples were acquired on LSR-II, LSRFortessa, or Fortessa X20 flow cytometers (BD), and analysis was performed with FlowJo software (Treestar).
Rane S., Hogan T., Seddon B, & Yates A.J. (2018). Age is not just a number: Naive T cells increase their ability to persist in the circulation over time. PLoS Biology, 16(4), e2003949.
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