Cd4 allophycocyanin

Manufactured by Thermo Fisher Scientific

The CD4-allophycocyanin is a fluorescent-labeled antibody that binds to the CD4 antigen. It is used to identify and quantify CD4-positive cells in flow cytometry applications.

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7 protocols using cd4 allophycocyanin

Comprehensive Immune Cell Phenotyping

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Antibodies used for flow cytometry analysis included: CD2-Tri-Color (TC); CD4-Phycoerythrin (PE) and CD4Allophycocyanin (APC); CD28-Fluorescein Isothiocyanate (FITC); CD8-TC; CD19-APC; CD45RA-APC; CD16-FITC from Life Technologies (Grand Island, NY); CD14-PE; CD27-PE; and IgM-FITC from BD Biosciences (San Jose, CA). Freshly thawed PBMCs from each visit were stained with three to five antibodies: T cells (CD2, CD4, CD8, CD45RA, and CD28); B cells (CD19, IgM, and CD27); NK cells (CD16). The data were collected on a BD FACSCalibur or BD FACSCanto II, and analyzed by Cell-Quest (BD Biosciences) and FlowJo. The gating strategies were presented in Additional file 1: Figure S1.

Lin Y., Kim J., Metter E.J., Nguyen H., Truong T., Lustig A., Ferrucci L, & Weng N.P. (2016). Changes in blood lymphocyte numbers with age in vivo and their association with the levels of cytokines/cytokine receptors. Immunity & Ageing : I & A, 13, 24.

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Characterizing HIV Infection in Primary CD4+ T Cells

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PBMCs were isolated from peripheral blood obtained via phlebotomy from
HIV-1–negative donors per an active, approved University of Utah IRB
protocol (#67637; principal investigator, A. M. Spivak). Total CD4⁺ T
cells were purified via negative magnetic bead selection using a total
CD4⁺ T cell–negative isolation kit (STEMCELL) according to
the manufacturer’s instructions. CD4⁺ T cells were cultured in
an RPMI 1640–based media supplemented with IL-2 (30 IU/ml) and activated
in vitro for 72 hours via exposure to antibodies to CD3 and CD28 conjugated to
magnetic beads (Invitrogen). Activated CD4⁺ T cells subsequently
underwent spin infection with an HIV-1_NL4–3 viral strain
pseudotyped with green fluorescent protein in place of env(multiplicity of infection of about 1.0 using a titer measured in SupT1 cells)
at 1200g for 2 hours at 37°C. CD4⁺ T cells were stained with
the following fluorophore-conjugated antibodies before flow cytometry
acquisition: CD3 (PE-Cy7; BioLegend), CD4 (allophycocyanin; Life Technologies),
CD32 (PE; Fun-2 clone; BioLegend), CD14 [Brilliant Violet (BV) 421; BioLegend],
and HLA-DR (BV 605; BD Biosciences). Flow cytometry was performed at 0, 24, 48,
and 72 hours relative to CD4⁺ T cell isolation, as well as 0, 24, and
48 hours after NL4–3 infection. Flow cytometry acquisition was performed
on a BD FACSCanto II instrument, and analysis was performed using FlowJo v10
software.

Abdel-Mohsen M., Kuri-Cervantes L., Grau-Exposito J., Spivak A.M., Nell R.A., Tomescu C., Vadrevu S.K., Giron L.B., Serra-Peinado C., Genescà M., Castellví J., Wu G., Del Rio Estrada P.M., González-Navarro M., Lynn K., King C.T., Vemula S., Cox K., Wan Y., Li Q., Mounzer K., Kostman J., Frank I., Paiardini M., Hazuda D., Reyes-Terán G., Richman D., Howell B., Tebas P., Martinez-Picado J., Planelles V., Buzon M.J., Betts M.R, & Montaner L.J. (2018). CD32 is expressed on cells with transcriptionally active HIV but does not enrich for HIV DNA in resting T cells. Science translational medicine, 10(437), eaar6759.

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Multiparametric Immune Cell Analysis

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All antibodies were purchased from commercial sources: CD3-phycoerythrin (PE), CD4-allophycocyanin (APC), CD4-peridinin-chlorophyll–protein complex (PerCP), CD19-PE, CD3-APC, CD3-fluorescein isothiocyanate (FITC), CD8-PE, NK1.1-APC, F4/80-PE, CD11c-PE, CD11c-APC, IL-4-PE, IFN-γ-APC, TNFα-FITC, CD69-APC, anti-CD3 Abs, and anti-CD28 Abs from eBioscience; Rabbit polyclonal GIT2 Abs, anti-phospho-PAK1/2 (Ser199/204 of PAK1 and Ser192/197 of PAK2 Abs, and anti-phospho-c-Cbl (Y774) Abs from Cell Signaling; Anti-phospho-Erk1/2 (E4) Abs, anti-PAKα, anti-Erk1/2 (K23) from Santa Cruz.

Hao Y.E., He D.F., Yin R.H., Chen H., Wang J., Wang S.X., Zhan Y.Q., Ge C.H., Li C.Y., Yu M, & Yang X.M. (2015). GIT2 deficiency attenuates concanavalin A-induced hepatitis in mice. FEBS Open Bio, 5, 688-704.

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Melanoma Immune Cell Profiling Protocol

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Melanoma tissues were digested with 1 U/ml collagenase (Sigma Aldrich, Italy), passed through 70-μm cell strainers and red blood cells (RBC) were lysed to prepare single cell suspensions. Cell samples were blocked with anti-mouse CD16/CD32 (eBioscience, San Diego, CA, USA). Antibodies against CD11c-FITC, CD11b-PeCy5.5, Gr1-PE or Gr1-allophycocyanin, CD3-PeCy5.5; CD8-allophycocyanin or CD8-PE; CD4-allophycocyanin; NK1.1-PE were obtained from eBioscience and BioLegend. Expression of FAP in melanoma tissue was also analyzed by flow cytometry by using an anti-FAP-FITC and expressed as percentage of positive cells. Data were acquired with a FACSCalibur flow cytometer (BD Biosciences).

Sorrentino C., Miele L., Porta A., Pinto A, & Morello S. (2016). Activation of the A2B adenosine receptor in B16 melanomas induces CXCL12 expression in FAP-positive tumor stromal cells, enhancing tumor progression. Oncotarget, 7(39), 64274-64288.

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Flow Cytometric Analysis of T Cell Viability

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Cells were washed once with PBS containing 1% BSA and incubated with live/dead near-IR fixable viability dye (Invitrogen), CD3 Brilliant Violet 650 (BioLegend), and CD4 Allophycocyanin (eBioscience) at 4 °C for 30 min. Cells were washed in PBS/BSA and fixed in PBS/BSA containing 1% paraformaldehyde prior to data acquisition. All samples were analyzed using a BD Fortessa (BD Biosciences) cell analyzer equipped with a High Throughput Sampler option. A minimum of 50,000 events were collected per sample at a flow rate of 2.5 μL/s with 50 μL mixing and 3 × 200 μL washes. All experimental conditions were performed in triplicate. FlowJo version 9.7.6 (TreeStar, Inc) was used for analysis. For downstream validation of significant hits, the above experiments above were repeated in triplicate on a minimum of 3 healthy donor CD4+ T cell populations.

Lucera M.B., Fleissner Z., Tabler C.O., Schlatzer D.M., Troyer Z, & Tilton J.C. (2017). HIV signaling through CD4 and CCR5 activates Rho family GTPases that are required for optimal infection of primary CD4+ T cells. Retrovirology, 14, 4.

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Phenotyping Melanoma Immune Cells

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Single cell suspensions were prepared from harvested melanoma tissues. Tissues were dissected and digested with collagenase 1U/ml, passed through 70-μm cell strainers and red blood cells (RBC) were lysed. Cell samples were pre-incubated with anti-mouse CD16/CD32 (eBioscience, San Diego, CA, USA) to block non-specific Fc-mediated interactions. Antibodies against CD11c-FITC, CD11b-PeCy5.5, Gr1-PE or Gr1-allophycocyanin, CD3-PeCy5.5; CD8-allophycocyanin or CD8-PE; CD4-allophycocyanin; NK1.1-PE were obtained from eBioscience and BioLegend. Data were acquired with a FACSCalibur flow cytometer (BD Biosciences).

Sorrentino C., Miele L., Porta A., Pinto A, & Morello S. (2015). Myeloid-derived suppressor cells contribute to A2B adenosine receptor-induced VEGF production and angiogenesis in a mouse melanoma model. Oncotarget, 6(29), 27478-27489.

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Flow Cytometry Analysis of Cell Fusion and Infection

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Cells were washed once with PBS containing 1% BSA and incubated with live/dead fixable yellow dead cell stain (Thermo Fisher), CD3 Brilliant Violet 650 (BioLegend), and CD4 Allophycocyanin (eBioscience) at 4 °C for 30 min. Cells were washed in PBS/BSA and fixed in PBS/BSA containing 1% paraformaldehyde prior to data acquisition. All samples were analyzed using an LSRII flow cytometer (Becton–Dickinson). A minimum of 50,000 events were collected per sample. FlowJo version 9.7.6 (TreeStar, Inc) was used for analysis. Flow cytometry plots were gated using fluorescence intensity. Summary data are presented as fusion (+) and infection (+) cells as a percentage of no drug controls. Post-infection efficiency represent the ratio of infection (+):fusion (+) cells, also expressed as a percentage of no drug controls. All summary data represent mean values with standard error of the mean unless stated otherwise. All differences with a p value of < 0.05 were considered statistically significant, correcting for multiple comparisons when appropriate. Statistical analyses were performed using student T-tests within GraphPad Prism v7.0.

Wiredja D.D., Tabler C.O., Schlatzer D.M., Li M., Chance M.R, & Tilton J.C. (2018). Global phosphoproteomics of CCR5-tropic HIV-1 signaling reveals reprogramming of cellular protein production pathways and identifies p70-S6K1 and MK2 as HIV-responsive kinases required for optimal infection of CD4+ T cells. Retrovirology, 15, 44.

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