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Cd4 percp cy5

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The CD4-PerCP-Cy5.5 is a fluorescent-labeled antibody that binds to the CD4 receptor on the surface of T helper cells. It is used for the identification and enumeration of CD4+ T cells in flow cytometry analysis.

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Lab products found in correlation

Cd3 apc cy7, by BD (10 mentions) Lsrii flow cytometer, by BD (9 mentions) Cd3 fitc, by BD (8 mentions) Cd8 apc h7, by BD (7 mentions) Cd25 pe, by BD (6 mentions) Cytofix cytoperm, by BD (6 mentions) Cd45ra pe cy7, by BD (5 mentions) Cd8 v500, by BD (5 mentions) Cd3 apc, by BD (5 mentions) Cd3 pacific blue, by BD (5 mentions) Cd3 pe cy7, by BD (5 mentions) Facsdiva software, by BD (5 mentions) Facscanto 2, by BD (5 mentions) Cd28 pe cy7, by BD (4 mentions) Cd3 apc h7, by BD (4 mentions) Cd8 apc cy7, by BD (4 mentions) Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (4 mentions) Cd20 apc cy7, by BD (4 mentions) Cd8 pe, by BD (4 mentions) Cd8 fitc, by BD (4 mentions)

Close spelling variants of this product

CD4-PerCP-Cy5.5 (RM4-5), PerCP-Cy5.5–conjugated anti-CD4 CD4-peridinin chlorophyll protein (PerCP).Cy5.5 (RM4-5; PerCP-Cy5.5-conjugated anti-CD4 (SK3; Anti-mouse CD4 PerCP-Cy5.5 Anti-CD4-PerCP-Cy5.5 CD4-PerCP-Cy5.5 mAb (clone 74-12-4, PerCP-Cy5.5–conjugated anti-mouse CD4 PerCP-Cy5.5 conjugated CD4 (RM4-5; FITC-anti-mouse CD4 or PerCP-Cy5.5-anti-mouse CD8 antibody

90 protocols using cd4 percp cy5

1

Tolerogenic Dendritic Cell Modulation of T-Cell Subsets

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After 5 days of culture, PBMC co-cultured with mature DC (mDC), VitD3-tolDC (100% VitD3-tolDC) and 50% VitD3-tolDC (50% VitD3-tolDC + 50% mDC) in the presence and absence of IFN-beta were harvested and stained using CD3-V450, CD4-PerCP-Cy5.5, CD45RA PE-Cy7, CCR7 PE, CD38 APC, CD8 APC-H7, HLA-DR V500 (BD Bioscience), CD183 AF488, CD196 BV605 and CD45 AF700 (Biolegend, San Diego, CA, USA) for T cell analysis; and CD4 PerCP-Cy5.5, CD25 PE, CCR4 PE-Cy7, CD127 AF647, CD45RO APC-H7, CD3 V450, HLA-DR V500 (BD Biosciences) and CD45 AF700 (Biolegend) for Treg analysis. Monoclonal antibodies were incubated for 20 min at room temperature and protected from the light. Samples were washed and a total of 50,000 CD3+ events were acquired on an LSR Fortessa flow cytometer (BD Biosciences). Both panels were analyzed using FACSDiva software (BD Biosciences). The gating strategy used to analyze the desired T cell subpopulations was previously reported [28 (link)].
Quirant-Sánchez B., Mansilla M.J., Navarro-Barriuso J., Presas-Rodríguez S., Teniente-Serra A., Fondelli F., Ramo-Tello C, & Martínez-Cáceres E. (2021). Combined Therapy of Vitamin D3-Tolerogenic Dendritic Cells and Interferon-β in a Preclinical Model of Multiple Sclerosis. Biomedicines, 9(12), 1758.
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2

Flow Cytometric Analysis of T Cell Subsets

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Blood samples were collected prior to the first administration of the treatment (baseline) and processed by a centralized laboratory within the first 24 h after their collection. Samples of whole blood were analyzed by flow cytometry to determine the percentage and absolute number of T lymphocyte subpopulations using the following combination of monoclonal antibodies per panel: CD3-V450, CD4 PerCP-Cy5.5, CD45RA PE-Cy7, CCR7 PE, CD38 APC, CD8 APC-H7, HLA-DR V500 (BD Biosciences), CD183 AF488, CD196 BV605, and CD45 AF700 (BioLegend, San Diego, CA, USA). The absolute cell number quantification was performed as previously reported [16 (link)]. Samples were acquired on a LSR II Fortessa flow cytometer (BD Biosciences, San José, CA, USA).
The following T cell subpopulations were analyzed: CD4+ naïve, CD4+ TCM, Th1CM, Th1Th17CM, Th2CM, Th17CM, CD4+ TEMRA, CD4+ TEM, Th1EM, Th1Th17EM, Th2EM, Th17EM, CD8+ naïve, CD8+ TCM, CD8+ TEMRA, CD8+ TEM, double positive (CD4+CD8+), and double negative (CD4−CD8−) T cells. Analysis was performed using the FACSDiva software (BD Biosciences). The gating strategy for the subpopulations analyzed in whole blood is shown in Figure 1 and previously described by Quirant-Sánchez et al. [22 (link)].
Quirant-Sánchez B., Presas-Rodriguez S., Mansilla M.J., Teniente-Serra A., Hervás-García J.V., Brieva L., Moral-Torres E., Cano A., Munteis E., Navarro-Barriuso J., Martínez-Cáceres E.M, & Ramo-Tello C. (2019). Th1Th17CM Lymphocyte Subpopulation as a Predictive Biomarker of Disease Activity in Multiple Sclerosis Patients under Dimethyl Fumarate or Fingolimod Treatment. Mediators of Inflammation, 2019, 8147803.
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3

Flow Cytometric Immune Profiling in Transplant

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Flow cytometric analysis was performed up to 3 times pre-transplant and serially post-transplant to characterize peripheral blood immune cell phenotypes. Total T cells and T cell subsets were quantified by complete blood cell count and flow cytometry. Fresh PBMCs were isolated by Ficoll density gradient centrifugation (BD Biosciences, Franklin Lakes, NJ). PBMCs were stained with the following mAbs: CD3 PacBlue, CD95 V450, CD3 Alexa 700, CD4 PerCP-Cy5.5, CD8 V500, CD28 PE-Cy7, CD25 PE-Cy7, IFNy PE-Cy7, CD28 APC, TNF APC, VLA-4 APC, CD11a PE, CD45RA FITC, CD40 FITC, CCR7 APC, CD20 APC (all BD Biosciences). PBMCs (1.5×106) were incubated with appropriately titered antibodies for 15min at 20°C and washed twice. Samples were acquired immediately on a BD LSR II multicolor flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, San Carlos, CA). For the stimulation assay, 1.5×106 PBMCs were cultured in RPMI 1640 (Corning cellgro, Manassas, VA) supplemented with 10% fetal bovine serum and stimulated with 10µM phorbol 12-myristate 13-acetate (PMA) and 200nM ionomycin (Sigma-Aldrich, St. Louis, MO), with 1ul/ml GolgiPlug protein transport inhibitor for 5 h, +/− IL-15 (10ng/mL). PBMCs were were processed with BD Cytofix/Cytoperm Plus kit (BD 555028) per the manufactures recommendation prior to data acquisition.
Mathews D., Wakwe W., Kim S., Lowe M., Breeden C., Roberts M., Farris A., Strobert E., Jenkins J., Larsen C., Ford M., Townsend R, & Adams A. (2017). Belatacept Resistant Rejection is Associated with CD28+ Memory CD8 T cells. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 17(9), 2285-2299.
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4

Flow Cytometric Analysis of Surface Markers

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To analyse expression of surface markers, MCs were incubated with fluorochrome‐labeled monoclonal antibodies for 30 min in the dark on ice and fluorescence intensity was measured using a LSR II flow cytometer (BD Biosciences). CD3 V500, CD3 Alexa Fluor 647, CD4 PerCP‐Cy5.5, CD8 APC‐H7, CD14 V500, CD14 Pe‐Cy7, CD27 BV510, CD43 FITC, CD45RA PeCy‐7, CD56 BV510 (BD Biosciences) and Siglec‐1 Alexa Fluor 647 (Sanbio) were used. Relative mean intensity fluorescence (MFI) of CD43 was calculated as follows: MFI stained/MFI unstained. GFP fluorescence was used to determine replication of (GFP)‐labeled RSV.
Jans J., Unger W.W., Vissers M., Ahout I.M., Schreurs I., Wickenhagen A., de Groot R., de Jonge M.I, & Ferwerda G. (2018). Siglec‐1 inhibits RSV‐induced interferon gamma production by adult T cells in contrast to newborn T cells. European Journal of Immunology, 48(4), 621-631.
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5

Isolation and Phenotypic Analysis of Naïve B Cells

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PBMCs were isolated from whole blood collected from family members and healthy donors by ficoll-histopaque gradient centrifugation. For phenotypic staining, the following monoclonal antibodies were used: CD3-APC-H7, CD4-PerCP-Cy5.5, CD38-PerCP-Cy5.5, CD10-PECF594, CD21-APC, IgG PeCy7, CD14-PerCP, CD123-PE, CD56-PeCy7, CD11c-APC, CD16-APC-H7 (BD Biosciences, San Diego, CA, USA), CD8-APC-EF780, CD27-APC-EF780 (eBioscience, San Diego, CA, USA), CD19-BV650, CD24-BV605 (Biolegend, San Diego, CA, USA) and IgA-PE (Miltenyi Biotech, Bergisch Gladbach, Germany). Naïve B cells were enriched by negative selection using B-cell isolation kit (Stemcell, Vancouver, BC, Canada). Naïve B-cell purity was verified by flow cytometry to 98% purity.
Ameratunga R., Koopmans W., Woon S.T., Leung E., Lehnert K., Slade C.A., Tempany J.C., Enders A., Steele R., Browett P., Hodgkin P.D, & Bryant V.L. (2017). Epistatic interactions between mutations of TACI (TNFRSF13B) and TCF3 result in a severe primary immunodeficiency disorder and systemic lupus erythematosus. Clinical & Translational Immunology, 6(10), e159-.
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6

In Vitro T Cell Suppression Assay

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In vitro suppression assays were carried out in RPMI/10% FCS in 96-well U-bottom plates (Corning, NY). Either naive splenocytes, CD3+ cells isolated from murine lymph nodes or leucocytes isolated from murine peripheral blood were utilized to perform three different sets of suppression assays. In all sets of experiments isolated cells were labeled with 5 μM CFSE (Molecular Probes), and activated in vitro with anti-CD3 and anti-CD28 beads (Invitrogen) according to the manufacturer’s instructions. Condition media of polarized macrophages was added to the culture. After 3-5 days, cells were acquired by BD LSR Fortessa or BD FACS Symphony and the proliferation of CFSE-labeled CD8+ T cells was assessed upon staining with the following anti-mouse monoclonal antibodies: CD3 APC-Cy7 (clone B241616); CD4 PerCP-Cy5.5 (clone B240053); CD8 APC (clone 53-6.7). Analysis of the data was performed by FlowJo software.
Di Mitri D., Mirenda M., Vasilevska J., Calcinotto A., Delaleu N., Revandkar A., Gil V., Boysen G., Losa M., Mosole S., Pasquini E., D’Antuono R., Masetti M., Zagato E., Chiorino G., Ostano P., Rinaldi A., Gnetti L., Graupera M., Martins Figueiredo Fonseca A.R., Pereira Mestre R., Waugh D., Barry S., De Bono J, & Alimonti A. (2019). Re-education of Tumor-Associated Macrophages by CXCR2 Blockade Drives Senescence and Tumor Inhibition in Advanced Prostate Cancer. Cell Reports, 28(8), 2156-2168.e5.
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7

Multiparameter Flow Cytometry for T-cell Immune Responses

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Multiparameter flow cytometry was used to determine T-cell immune responses using peptide stimulated PBMC as previously described.23 (link) One million PBMC were stained for each condition (DMSO, PMA/Ionomycin, or peptide(s)) for 10–14 hours. PBMC were stained with the following antibodies: Live/Dead Yellow (Invitrogen®, Catalog #:L34959), CD3-APC (BD Biosciences, clone SP34-2, Catalog #: 551916), CD4-PerCP Cy5.5 (BD BioSciences, clone L200, Catalog #: 551980), CD8 APC-Cy7 (BD Biosciences, clone RPA-T8, Catalog #: 55760), IFNγ-BV650 (BioLegend, clone 4S.B3, Catalog #: 502537), IL-2-PE (BioLegend, clone MQ1-17H12, Catalog #: 500307), TNFα-PECy7 (BD Biosciences, clone Mab11, Catalog #: 557647), CD107a-FITC (BD Biosciences, clone H4A3, Catalog #: 555800). Cells were fixed in 1% paraformaldehyde and acquired using an LSR II flow cytometer (BD Biosciences) and the data analyzed using FlowJo software (Tree Star, Inc.). Samples were considered positive if peptide-specific responses were at least twice that of the negative control plus at least 0.01% after background subtraction.
Munson P., Liu Y., Bratt D., Fuller J.T., Hu X., Pavlakis G.N., Felber B.K., Mullins J.I, & Fuller D.H. (2018). Therapeutic conserved elements (CE) DNA vaccine induces strong T-cell responses against highly conserved viral sequences during simian-human immunodeficiency virus infection. Human Vaccines & Immunotherapeutics, 14(7), 1820-1831.
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8

Flow Cytometry of Immune Cell Subsets

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The following fluorochrome conjugated antihuman monoclonal antibodies (MoAbs) were used for flow cytometry studies: ICOSBV421, ICOSAlexa488, CD40LBV605, CD69BV650, HLA-DRFITC, CD38APCCy7, and TNF-αAPCCy7 from BioLegend (San Diego, CA); CD3BUV395, CD4PerCPCy5.5, CD8Alexa-Fluor700, CCR7PECF594, IL-2BV711, CXCR5Alexa647, IFNγPE-Cy7, PD1BV650, CD45ROAPCH7, CD21PECy5, CD27PerCPCy5.5, IgDFITC, CD10PECy7, and CD20Alexa700 from BD Bioscience (San Jose, CA); IL-21PE, CD27PECy5, and IL-17Alexa488 from e-Biosciences (San Diego, CA); and CD45ROPE-TexasRed from Beckman Coulter (Fullerton, CA). Live/Dead Fixable Aqua Dead Cell Stain Kit and CellTrace Violet Cell Proliferation Kit were from ThermoFisher (Boston, MA). Recombinant human IL-21 (Cat #8879-IL-010) from R&D systems, purified antihuman IL-2 (Cat #3440-ON-500) and antihuman-IL-21 (Cat #MT216G/21.3m) from Mabtech, and antihumanTNF-α (Cat #502922) from BioLegend were used. Samples were acquired on a BD LSRFortessa (BD Biosciences, CA) flow cytometer and analyzed by FlowJo V10 (TreeStar, Inc).
Pallikkuth S., de Armas L.R., Rinaldi S., George V.K., Pan L., Arheart K.L., Pahwa R, & Pahwa S. (2019). Dysfunctional peripheral T follicular helper cells dominate in people with impaired influenza vaccine responses: Results from the FLORAH study. PLoS Biology, 17(5), e3000257.
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9

Comprehensive Immune Cell Profiling Post-Transplantation

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Analysis of circulating immune cell phenotypes was performed both prior to transplant and at regular intervals following transplantation. Cell frequencies from flow cytometric analysis were combined with complete blood counts to calculate total numbers of circulating T cells and various T cell subsets. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density centrifugation (BD Biosciences, Franklin Lakes, NJ) within 6 hours of phlebotomy. PBMCs (1.5×10^6) were then incubated with antibody mixtures at the appropriate titer for 15 minutes and washed twice. For assessment of intracellular markers, cells were fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) according to the manufacturer’s direction following surface staining. Flow cytometric data was acquired immediately using a BD LSR II multicolor flow cytometer (BD Biosciences). All flow data was analyzed using FlowJo (Tree Star, San Carlos, CA).
Surface markers were stained with the following monoclonal antibodies (mAbs): CD3 PacBlue, CD3 APC-Cy7, CD4 PerCP-Cy5.5, CD8 V500, CD28 PE-Cy7, CD127 PE-Cy7, PD-1 APC, LFA-1 APC, CD20 APC-Cy7 (all BD Biosciences), CD95 PacBlue, CD69 FITC (Invitrogen, Grand Island, NY), and CD25 PE (Miltenyi Biotech, San Diego, CA). Intracellular staining for FoxP3 was performed using FoxP3 Alexa488 (Biolegend, San Diego, CA).
Anderson D.J., Lo D.J., Leopardi F., Song M., Strobert E.A., Jenkins J.B., Larsen C.P, & Kirk A.D. (2019). Corticosteroids and Methotrexate as Adjuvants to Costimulation Blockade in Nonhuman Primate Renal Transplantation. Clinical transplantation, 33(6), e13568.
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10

Multifaceted Cellular Analysis by FACS

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Analysis of samples was performed on a LSR2 and Fluorescence-activated Cell Sorting(FACS) on an ARIA or Fusion-instrument (BD Biosciences, San Jose, CA, USA). Single cells were gated on the basis of FSC-Height(FSC-H) and FSC-Area(FSC-A), and live cells were gated out using Propidium Iodide(PI) or the LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (ThermoFisher Scientific, Waltham, MA, USA). Antibodies were obtained from BioLegend (San Diego, CA, USA): CD1a PB, CD3 Pe-Cy7, CD3 APC-Cy7, CD3 BV421, CD4 PerCp-Cy5.5, CD5 PE, CD8 Amcyan, CD8 APC-Cy7, CD14 PB, CD19-APC, CD27 APC-Cy7, CD33 PE, CD34 PerCp-Cy5.5, CD34 APC, CD45 Amcyan, CD45 Pe-Cy7, CD56 BV421, CD69 Pe-Cy7, CD137 PE, interferon-γ PE, IL-2 PB, TCRαβ-PE, and TCRγδ APC; BD Biosciences (San Jose, CA, USA): CD7-FITC; ThermoFisher Scientific (Waltham, MA, USA): CellTrace™ Violet Cell Proliferation Kit; and SCBT (Dallas, TX, USA): WASp-AF647.
Pille M., Avila J., Sanchez G.S., Goetgeluk G., De Munter S., Jansen H., Billiet L., Weening K., Xue H., Bonte S., Ingels J., De Cock L., Pascal E., Deseins L., Kerre T., Taghon T., Leclercq G., Vermijlen D., Davis B, & Vandekerckhove B. (2023). The Wiskott–Aldrich syndrome protein is required for positive selection during T-cell lineage differentiation. Frontiers in Immunology, 14, 1188099.
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