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Cd4 percp

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CD4-PerCP is a fluorescently-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 applications.

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Lab products found in correlation

Cd25 apc, by BioLegend (6 mentions) Pd 1 pe, by BioLegend (5 mentions) Cd25 fitc, by BioLegend (4 mentions) Cd25 fitc, by Thermo Fisher Scientific (3 mentions) Cd8 fitc, by BioLegend (3 mentions) Cd3 fitc, by BD (3 mentions) Cd4 pe, by BioLegend (3 mentions) Cd8 fitc, by BD (3 mentions) Alexa 488 cd4, by BioLegend (2 mentions) Il 17a pe, by BioLegend (2 mentions) Accuri benchtop c6 cytometer, by FlowJo (2 mentions) Fix perm intracellular staining buffers, by Thermo Fisher Scientific (2 mentions) Foxp3 pe, by Thermo Fisher Scientific (2 mentions) Alexa647 foxp3, by BioLegend (2 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions) Cd11b apc, by Thermo Fisher Scientific (2 mentions) Cd11c apc, by BioLegend (2 mentions) Cd40 pe, by BioLegend (2 mentions) Efluor 506, by Thermo Fisher Scientific (2 mentions) Ifn γ fitc, by Thermo Fisher Scientific (2 mentions)

30 protocols using cd4 percp

1

Multiparametric Flow Cytometry for T-Cell Analysis

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CBMCs and maternal PBMCs were thawed, aliquoted at 1 × 106 cells, cells were washed, stained, fixed, and permeabilized, as per the manufacturer’s instructions (FoxP3 Fix/Perm kit; eBiosciences) using standard protocols using the following antibodies: APC/Cy7 CD3 (clone OKT3), PerCP CD4 (clone RPA-T4), Brilliant Violet 421 CD25 (clone BC96), Brilliant Violet 650 CD127 (clone A019D5), Brilliant Violet 605 CD45RO (clone UCHL1), FITC CCR7 (clone G043H7), Brilliant Violet 510 CD8 (clone SK1), Brilliant Violet 510 CD14 (clone M5E2), Brilliant Violet 510 CD19 (clone HIB19, BioLegend), FITC Ki67 (clone Ki67, BD Pharmingen), PE FoxP3 (clone PCH101, eBioscience), and LIVE/DEAD aqua amine (Invitrogen). Flow cytometry data were collected on an LSR II four-laser flow cytometer (BD) with FACSDiva software. Color compensation was performed using compensation beads. Fluorescence-minus-one samples were used to define negative and positive populations. Cellular profiles were gated on live, single-cell, dump negative (CD14, CD19, CD8), CD3 + lymphocytes.
Prahl M., Odorizzi P., Gingrich D., Muhindo M., McIntyre T., Budker R., Jagannathan P., Farrington L., Nalubega M., Nankya F., Sikyomu E., Musinguzi K., Naluwu K., Auma A., Kakuru A., Kamya M.R., Dorsey G., Aweeka F, & Feeney M.E. (2021). Exposure to pesticides in utero impacts the fetal immune system and response to vaccination in infancy. Nature Communications, 12, 132.
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2

Multiparametric Flow Cytometry of Prostate

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Single cell suspensions were generated from whole prostate tissues combining all prostate lobes (as above in bacterial colonization) and iliac lymph node tissues by homogenization and filtration. Cells were washed in FACS buffer (2% FCS (Hyclone) in PBS (Gibco)) and staining performed using conjugated antibodies: PerCp-CD4, Alexa-488-CD4, and PE-IL17A (Biolegend), and PC-CD11b, FITC-CD273, PE-CD274, CD3, CD8, and IL4 (eBioscience). Samples were run on an Accuri-benchtop-C6 cytometer and analyzed using FlowJo™. For all analyses, unless otherwise stated, samples were gated on cell populations based on lymphocyte or monocyte size, as assessed by SSC and FSC, followed by gating for CD11b, CD3, or CD4. Intracellular staining was performed using IC fixation buffer (eBioscience) and Permeabilization Buffer (eBioscience). Staining was performed for 1 hour at 25C followed by washing in FACS buffer.
Murphy S.F., Anker J.F., Mazur D.J., Hall C., Schaeffer A.J, & Thumbikat P. (2018). Role of gram-positive bacteria in chronic pelvic pain syndrome (CPPS).. The Prostate, 79(2), 160-167.
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3

Multiparameter Flow Cytometry Profiling

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Flow cytometry was performed on single cell suspensions using these mouse antibodies; PerCp-CD4, Alexa-488-CD4, Alexa-647-FoxP3, PE-IL17A, FITC-CD25, PE-PD1 (Biolegend), PE-FoxP3, PC-IL17, FITC-CD25, APC-CD11b, FITC-PDL2, PE-PDL1 (eBiosciences). Flow cytometry was run on an Accuri benchtop C6 cytometer and analysed using FlowJo™ software. For all analyses unless otherwise stated, samples were gated on lymphocyte populations based on size, as assessed by SSC and FSC, followed by gating for CD4 positivity. Intracellular staining was performed by fixation and permeabilization using eBioscience Fix-Perm Intracellular staining buffers (Cat. Num 8222-49 and 8333-56). Staining was performed for 1 hour at room temperature followed by washing in FACS buffer (2% FCS (Hyclone), in PBS (Gibco)) Analyses were performed using FlowJo™ and data statistically tested using GraphPad Prism™ software, with tests described in respective figure legends.
Murphy S.F., Schaeffer A.J., Done J.D., Quick M.L., Acar U, & Thumbikat P. (2017). Commensal bacterial modulation of the host immune response to ameliorate pain in a murine model of chronic prostatitis. Pain, 158(8), 1517-1527.
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4

Comprehensive T-cell Immunophenotyping Protocol

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Flow cytometric analyses of T-cell markers was performed using the following antibodies, mouse samples (PerCp-CD4 (Biolegend), FITC-CD25, PE-FoxP3, FITC-IFNg, APC-IL17 (eBiosciences), or human samples (PE-IL17, Alexa647-FoxP3, (Biolegend), PerCpCy5.5-CD4, FITC-IFN-g (eBiosciences) on an Accuri benchtop C6 cytometer. For all analyses unless otherwise stated, samples were gated on lymphocyte populations based on size, as assessed by SSC and FSC, followed by gating for CD4 positivity. Intracellular staining was performed by fixation and permeabilization using eBioscience Fix-Perm Intracellular staining buffers (Cat. Num 8222–49 and 8333–56). Staining was performed for 1 hour at room temperature followed by washing in FACS buffer (2% FCS (Hyclone), in PBS (Gibco)) Analyses were performed using FlowJo and data statistically tested using GraphPad Prism software, with tests described in respective figure legends.
Murphy S.F., Schaeffer A.J., Done J., Wong L., Bell-Cohn A., Roman K., Cashy J., Ohlhausen M, & Thumbikat P. (2015). IL17 Mediates Pelvic Pain in Experimental Autoimmune Prostatitis (EAP). PLoS ONE, 10(5), e0125623.
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5

Multiparameter Flow Cytometry Analysis

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The antibodies used were as follows: CD19-PECy7 (1D3), CD8-APC (53-6.7), Ly6C-FITC (AL-21), CD68-PE (FA11), CD44-FITc (IM7), CD45-PerCP (30-F11), CD69-PE (H1.2F3), CXCR5-ef450 (SPRCL5), CD138-APC (281-2), CD21-PE (7G6), CD275-PE (HK5.3), CD122-APC (TM-b1), CD11b-PB (M1/70.15), IFNγ-eFluor 450 (XMG1.2), CD4-PerCP (L3T4), F4/80-APC-eFluor780 (BM8), Foxp3-PE (MF23), CD3-APC-CY7/eFluor780 (17A2), Ly-6G-PE (1A8), from eBioscience (San Diego, CA) and anti-mouse CD16/CD32 (The Lymphocyte Culture Centre, UVA, Charlottesville, VA). For some staining, we used: PerCP-CD4, biotinylated-CXCR5, followed by APC-streptavidin, or FITC-GL-7, PE-FAS and PerCP-B220 staining (all from Biolegend). To distinguish between live and dead cells, Viability Live Dead-ef650 (eBioscience, San Diego, CA) was used. Anti–CD3 and –CD28 were utilized for in vitro assays (eBioscience, San Diego, CA). Recombinant proteins used were as followed: mouse TGFβ and IL-2 was purchased from PeproTech (Rocky Hill, NJ). LPS was purchased through Sigma-Aldrich (St. Louis, MO).
Taghavie-Moghadam P.L., Wassem T.C., Hattler J., Glenn L.M., Dobrian A.D., Kaplan M.H., Yang Y., Nurieva R., Nadler J.L, & Galkina E.V. (2017). STAT4 regulates CD8+Treg/Tfh cell axis and promotes atherogenesis in insulin-resistant Ldlr−/− mice. Journal of immunology (Baltimore, Md. : 1950), 199(10), 3453-3465.
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6

Phenotyping Immune Cell Subsets

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DC (2 × 106 cells) and spleen lymphocytes (2 × 106 cells) were incubated with APC-CD11c (BioLegend, cat.117310), PE-CD80 (BioLegend, cat.104707), PE-CD86 (BioLegend, cat.105007), PE-CD40 (BioLegend, cat.124609), FITC-I-A/I-E (BioLegend, cat.), PE-CD3 (BioLegend, cat.100205), PerCP-CD4 (BioLegend, cat.100431), and FITC-CD8 (BioLegend, cat.100705) antibodies and the proportion of positive cells was analyzed by flow cytometry.
Li Y., Yang X., Zhao R., Xiu Z., Li S., Li Y., Song G., Ge C., Fang J., Han J., Zhu Y, & Li Y. (2023). Human adenovirus type 7 subunit vaccine induces dendritic cell maturation through the TLR4/NF-κB pathway is highly immunogenic. Frontiers in Cellular and Infection Microbiology, 13, 1117230.
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7

Characterization of Activated T Cells in RA

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Previously frozen PBMCs from RA subjects were cultured at 5 × 106 total cells/well within a 24 well plate in RPMI-1640 + 10% pooled human serum with 10 µg/ml of peptide. IL-2 (Novartis) was added at 325 IU/ml on day 6. After 14 days cells were stained for expression of CD25 FITC (BD), CD4 PerCP (BioLegend), Annexin V APC (BD), CD14 APC (BioLegend), CD19 APC (BioLegend), and tetramer before being run on a FACSCalibur. The data was analyzed by FlowJo software version 9.6.2 (Tree star).
James E., Rieck M., Pieper J., Gebe J.A., Yue B.B., Tatum M., Peda M., Sandin C., Klareskog L., Malmström V, & Buckner J.H. (2014). Citrulline specific Th1 cells are increased in rheumatoid arthritis and their frequency is influenced by disease duration and therapy. Arthritis & rheumatology (Hoboken, N.J.), 66(7), 1712-1722.
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8

Flow Cytometry Immunophenotyping Protocol

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For flow cytometry, the following fluorochrome-conjugated monoclonal antibodies were used. BD Biosciences: HLA-DR-APC (Clone: G46-6), CD86-FITC (Clone: FUN-1), CD80-PE (Clone: L307.4), CD54-APC (Clone: HA58), CD25-FITC (Clone: M-A251), CD127-BV421 (clone HIL-7R-M21), IFN-γ-FITC (Clone: 4S.B3), IL-4-PE (Clone: MP4-25D2); eBioscience: FoxP3-APC (Clone: 236A/E7), IL-17A-PE (Clone: Ebio64cap17); Beckman Coulter: CD40-PE (Clone: MAB89); Biolegend: CD4-PerCP (Clone: SK3). Cell viability was detected using the fixable viability dye eFluor 506 (eBioscience).
Antigen affinity-purified polyclonal anti-human TLR4 goat IgG was purchased from R&D systems. Cytokines (recombinant human granulocyte-macrophage colony-stimulating factor and IL-4), MicroBeads (CD14 and CD4) and cell purification units were obtained from Miltenyi Biotec. Protein-A agarose beads were from Cell Signalling Technology, and E. coli 055:B5 LPS and Polymyxin B-conjugated agarose beads were from Sigma-Aldrich. TLR4 signaling inhibitor CLI-095 and CpG ODN 2006 were procured from InvivoGen.
Mukherjee S., Karnam A., Das M., Babu S.P, & Bayry J. (2019). Wuchereria bancrofti filaria activates human dendritic cells and polarizes T helper 1 and regulatory T cells via toll-like receptor 4. Communications Biology, 2, 169.
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9

Multiparametric Flow Cytometry Analysis

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CD45RA-FITC and CD45RO-PE antibodies were obtained from BD Biosciences (San Diego, CA). CD38-APC and CD4-PerCP antibodies were obtained from BioLegend (San Diego, CA). Fresh peripheral blood mononuclear cells were stained using antibodies cocktail in 20 min at room temperature, and then, red cells were lysed in 15 min with Versalyse (Beckman Coulter, Brea, CA). Flow cytometry and data analysis were, respectively, performed on a BD CANTO II (BD Biosciences, San Diego, CA).
Ding Y., Pu C., Zhang X., Tang G., Zhang F, & Yu G. (2023). Identification of Potential Diagnostic Genes of HIV-Infected Immunological Non-Responders on Bioinformatics Analysis. Journal of Inflammation Research, 16, 1555-1570.
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10

Multicolor Flow Cytometry Analysis of Adipose Cells

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Multicolor flow cytometry measurements were performed and analyzed as described previously [23 (link),25 (link)]. Briefly, cells isolated from the SVF, which were obtained following collagenase digestion and lysis of red blood cells, were stained for flow cytometry using two antibody cocktails. Cocktail 1 included CD45-PE-Cy7 (BD 557748), CD3-fitc (BD 561807), CD19-fitc (BD 555412), CD56-fitc (BD 562794), CD66b-fitc (BD 555724), CD11b-BV421 (Biologend 301324), and CD11c-APC-Cy7 (Biolegend 337218). Cocktail 2 included CD45-PE-Cy7 (BD 557748), CD3-V500 (BD 561416), CD4-PerCP (Biolegend 300528), CD8 APC-H7 (BD 641400), CD19-BV421 (Biolegend 302234), and CD56-APC (Biolegend 318310). Samples were measured with a FACS-Canto II (BD Biosciences, Gurugram, India). Results were analyzed with FACSdiva (BD Biosciences) and FlowJo software version 10. Since weight of the adipose tissues was unavailable, data are expressed as % of cells relative to total cells (based on forward and side scatter plot).
De Hert E., Verboven K., Wouters K., Jocken J.W, & De Meester I. (2022). Prolyl Carboxypeptidase Activity Is Present in Human Adipose Tissue and Is Elevated in Serum of Obese Men with Type 2 Diabetes. International Journal of Molecular Sciences, 23(21), 13529.
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