Cd4 efluor450

Manufactured by Thermo Fisher Scientific

Sourced in United States

The CD4-eFluor450 is a fluorescently-labeled antibody for the detection of CD4-positive cells. It is designed for use in flow cytometry applications to identify and quantify CD4-expressing cells within a sample.

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Lab products found in correlation

Lsrii flow cytometer, by BD (6 mentions) Ccr7 pe cy7, by BD (4 mentions) Foxp3 pe, by Thermo Fisher Scientific (3 mentions) Cd8 apc h7, by BD (3 mentions) Cd3 efluor 605, by Thermo Fisher Scientific (2 mentions) Cd27 apc efluor780, by Thermo Fisher Scientific (2 mentions) Cd161 pe, by Miltenyi Biotec (2 mentions) Multitest trucount method, by BD (2 mentions) Facscanto clinical software, by BD (2 mentions) Cd8 percp, by BD (2 mentions) Cd45ro fitc, by BD (2 mentions) Cd8 apc efluor 780, by Thermo Fisher Scientific (2 mentions) Cd161 apc, by Miltenyi Biotec (2 mentions) Kaluza analysis software, by Beckman Coulter (2 mentions) Cd4 percp, by BD (2 mentions) Facscanto 2, by BD (2 mentions) Cd69 pe, by Thermo Fisher Scientific (2 mentions) Cd45 percp cy5, by Thermo Fisher Scientific (2 mentions) Cd3 apc efluor780, by Thermo Fisher Scientific (2 mentions) Fcs express software, by De Novo Software (2 mentions)

Close spelling variants of this product

Anti-mouse CD4-eFluor450 (3 citations) EFluor450-anti-CD4 (2 citations)

23 protocols using cd4 efluor450

Comprehensive Immune Cell Profiling by Flow Cytometry

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Peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation with Lymphoprep (Axis-Shield). PBMC or whole blood samples were stained with the following fluorochrome-conjugated monoclonal antibodies: CD3-efluor605, CD4-efluor450, CD27-APC-efluor780, HLA-DR-efluor780, CD45RA-efluor605, FOXP3-PE (eBioscience), CD4-APC-H7, CD8-Percp, CD8-PE-Cy7, CD31-AF647, CD45RO-FITC, CD45RO-PE-Cy7, CCR7-PE-Cy7, Ki-67-Percp-cy5.5, CTLA-4-BV421 (BD Biosciences), PD-1-PE, CD28-AF700 (Biolegend), and CD161-PE (Miltenyi Biotec). Intracellular staining for FOXP3, Helios, Ki-67, and CTLA-4 was performed after cells were permeabilized with a FOXP3 staining buffer set according to instructions of the manufacturer (eBioscience). Whole blood samples were treated with BD lysing solution according to the instructions of the manufacturer (BD Biosciences). Stained samples were analyzed on a LSR-II flow cytometer (BD Biosciences). Analysis was performed with Kaluza Flow Analysis Software (Beckman Coulter). Absolute numbers of CD3+ T cells, CD4+ T cells, CD8+ T cells, B cells, and NK cells were determined according to the MultiTest TruCount method (BD Biosciences), as described by the manufacturer. TruCount samples were measured on a FACSCanto-II (BD Biosciences) and analyzed with FACSCanto Clinical Software (BD Biosciences).

van den Brom R.R., van der Geest K.S., Brouwer E., Hospers G.A, & Boots A.M. (2018). Enhanced expression of PD-1 and other activation markers by CD4+ T cells of young but not old patients with metastatic melanoma. Cancer Immunology, Immunotherapy, 67(6), 925-933.

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Evaluating T-cell responses to Plasmodium falciparum

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PBMCs from day 0, 42, and 70 were assessed for P. falciparum derived peptide-specific activation induced proliferation. PBMCs were washed and cultured overnight. They were then suspended in RPMI media without any supplements at a cell density of 10×10⁶ live cells/ml and stained with 5 μM of CFDA SE (Molecular Probes, Eugene, OR). Following staining, cells were washed and suspended in HR10 at a cell density of 2×10⁶ cells/ml and plated into 96 well tissue culture plates (Corning, Corning, NY) at cell density of 2×10⁵ cells/well. Cells were stimulated either with individual P. falciparum derived HLA-DR-specific peptides (10 μg/ml), pan-DR epitope, PADRE (10 μg/ml), candida antigen (Greer Lab, 10 μg/ml) or the superantigen SEB (Toxin Technology, Sarasota, FL, 10 ng/ml). Peptides included in this analysis are outlined in Supplemental Table 5. Unstimulated cells or those stimulated with DMSO alone served as negative controls. Plates were incubated in 37°C, 5% CO₂ incubator for 6 days followed by staining with an antibody cocktail consisting of anti-human CD3-Alexa Fluor 700 (BD Biosciences), CD4-eFluor450 (eBioscience, San Diego, CA) and CD8-APC cy7 (BD Biosciences). Data was acquired on BD LSR-II flow cytometer and analyzed using Flowjo software (Treestar, Ashland, OR). The percentage of CD3+CD4+ and CD3+CD8+ cells in the proliferating gate were calculated.

Spearman P., Mulligan M., Anderson E.J., Shane A.L., Stephens K., Gibson T., Hartwell B., Hannaman D., Watson N.L, & Singh K. (2016). A Phase 1, Randomized, Controlled Dose-Escalation Study of EP-1300 Polyepitope DNA Vaccine Against Plasmodium Falciparum Malaria Administered via Electroporation. Vaccine, 34(46), 5571-5578.

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Sorting CD4+ T cell subsets

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CD4⁺ T cells were sorted into T_CM and T_EM subsets using a BD FACS Aria (BD Biosciences, San Jose, CA) run by BD FACS DIVA software. T_CM were defined as live, singlet, CD3⁺CD4⁺CD27⁺CD45RO⁺CCR7⁺ cells. T_EM were defined as live, singlet, CD3⁺CD4⁺CD27⁻CD45RO^+/dimCCR7⁻ cells, T_SCM were defined as live, singlet, CD3+CD4+CD27+CD45O-CCR7+CD95+ cells as previously described and as demonstrated in Figure 2D[23] (link). Cryopreserved PBMCs were thawed and stained with predetermined optimal concentrations of the following markers: Aqua Amine Reactive Dye (Invitrogen), CD3 AL700, CD8 PERCPCY5.5, CCR5 PE, CCR7 PE-CY7, CD95 PE-CY5 and Ki67 FITC (BD Pharmingen), CD4 eFluor450, CD27 APC-eFluor780 (eBiosciences, San Diego, CA), CD45RO ECD (Beckman Coulter, Chaska, MN).

Klatt N.R., Bosinger S.E., Peck M., Richert-Spuhler L.E., Heigele A., Gile J.P., Patel N., Taaffe J., Julg B., Camerini D., Torti C., Martin J.N., Deeks S.G., Sinclair E., Hecht F.M., Lederman M.M., Paiardini M., Kirchhoff F., Brenchley J.M., Hunt P.W, & Silvestri G. (2014). Limited HIV Infection of Central Memory and Stem Cell Memory CD4+ T Cells Is Associated with Lack of Progression in Viremic Individuals. PLoS Pathogens, 10(8), e1004345.

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Isolation and Characterization of Mouse Immune Cells

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Mouse splenocytes were grown in the conditions outlined above and spun down at 450 ×g at 4 °C for 5 min to pellet the cells. The supernatant was removed and the cells were re-suspended in 2 mL of FACS buffer (PBS + 2% FBS). Cells were then spun at 450 ×g at 4 °C for 5 min. The supernatant was again removed, and the cells were re-suspended in 200 μL of FACS buffer +2 μL of mouse CD16/CD32 blocking antibody (BD Biosciences, RRID: AB_2740348) and incubated on ice for 10 min. After incubation, 2 μL each of mouse CD45-PE, CD4-eFluor 450, and CD8-APC-eFluor 780 (eBiosciences, RRID: AB_469717, AB_467067, AB_11180008) were added, and the cells were incubated on ice for 30 min. All cells that were CD45+ and CD4+ or CD45+ and CD8+ were sorted by flow cytometry analysis and collected for molecular analysis.

McGuire M.H., Herbrich S.M., Dasari S.K., Wu S.Y., Wang Y., Rupaimoole R., Lopez-Berestein G., Baggerly K.A, & Sood A.K. (2019). Pan-cancer genomic analysis links 3’UTR DNA methylation with increased gene expression in T cells. EBioMedicine, 43, 127-137.

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Flow Cytometry Analysis of Lymphocytes

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Flow cytometry was performed using a BD Biosciences LSRFortessa. For direct ex vivo staining of lymphocytes, 1×10⁶ to 2×10⁶ cells were resuspended in 100 mL of FACS buffer (PBS þ 1% Fetalclone III), and Fc receptors were blocked for 10 to 15 minutes at 4 C with 1 µg/sample of 2.4G2 mAb (CD16 and CD32 blockade) and 1 µg/sample of mouse IgG (ThermoPierce). Cells were fluorescently labeled with antibodies for 30 minutes at 4 C, washed, and resuspended in fixation buffer (2% formaldehyde in PBS), or intracellularly stained according to the manufacturer's protocol (eBiosciences). The following fluorescently labeled anti- mouse mAbs were used for flow cytometry: IFNγ -FITC, IFNγ-PE, CD44-PerCP-Cy5.5, CD4-APC, CD8a-APC (BioLegend); CD62L- FITC, FoxP3-PE, TNFα-PerCP-eFluor 710, CD45-eFluor 450, CD4-eFluor450, CD62L-eFluor 450, CD4-APC-eFluor 780, and CD8a-APC-eFluor 780 (eBioscience); active caspase-3-PE (BD Biosciences). Flow cytometry data were analyzed with FCS Express 4 (De Novo Software).

Higuchi T., Flies D.B., Marjon N.A., Mantia-Smaldone G., Ronner L., Gimotty P.A, & Adams S.F. (2015). CTLA-4 Blockade Synergizes Therapeutically with PARP Inhibition in BRCA1-Deficient Ovarian Cancer. Cancer immunology research, 3(11), 1257-1268.

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Immunophenotyping of T Cell Subsets

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Routine assessment of differential blood counts was used to define the engraftment of neutrophil leukocytes and reconstitution of whole lymphocytes. Samples for immunophenotyping were taken on day 60, day 120 and day 180 following transplantation. Twenty healthy stem cell donors were analyzed to define normal ranges (controls).
CD4⁺ and CD8⁺ T cells were characterized according to the expression of CCR7 and CD45RA as naïve (CCR7⁺CD45RA⁺), central memory (CM, CCR7⁺CD45RA⁻), effector memory (EM, CCR7⁻CD45RA⁻), and terminally differentiated effector memory (TEMRA, CCR7⁻ CD45RA⁺) T cells.^{17 (link)} Staining was performed using the following antibodies as previously described:^{18 (link)} CD45-V500, CD3-PerCP-Cy5.5, CD8-APCH7, CCR7-FITC, CD45RAPE (all BD Biosciences, San Jose, CA, USA) and CD4-eFluor450 (eBioscience, San Diego, CA, USA) (Online Supplementary Figure S1). Immunophenotyping was performed using a BD FACSCanto II (BD Bioscience, San Jose, CA, USA).

Link-Rachner C.S., Eugster A., Rücker-Braun E., Heidenreich F., Oelschlägel U., Dahl A., Klesse C., Kuhn M., Middeke J.M., Bornhäuser M., Bonifacio E, & Schetelig J. (2019). T-cell receptor-α repertoire of CD8+ T cells following allogeneic stem cell transplantation using next-generation sequencing. Haematologica, 104(3), 622-631.

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Multiparametric Flow Cytometry Analysis

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Blood samples (EDTA) were stained with the following monoclonal antibodies: CD3-eFluor605, CD4-eFluor450, TCRγδ-PE (eBioscience), CD45RO-FITC, CCR7-PE-Cy7, CD4-PerCP, CD8-PerCP, CD8-APC-H7, DNAX accessory molecule-1 (DNAM-1)-FITC (BD), CD161-PE, CD161-APC (Miltenyi), 2B4-PE, NKG2D-PE-Cy7, TCR-Vα7.2-FITC, TCR-Vα24-Jα18-FITC (Biolegend), TCR-Vβ11-PE (Beckman Coulter), and KLRG1-FITC (generous gift from H. Pirchner). An overview of antibody panels is shown in Table S1 in Supplementary Material. Samples were subsequently treated with BD Lysing Solution (BD Biosciences) according to instructions of the manufacturer. Samples were measured on a LSR-II flow cytometer (BD) and analyzed with Kaluza Analysis Software (Beckman Coulter). In addition, the absolute numbers of circulating lymphocyte subsets were determined according to the MultiTest TruCount method (BD), as described by the manufacturer. Data were acquired on a FACSCanto-II flow cytometer (BD) and analyzed with FACSCanto Clinical Software (BD). The number of events for a particular T cell population needed to be more than 100 to allow for subsequent analysis of cellular markers, cytokines, and cytotoxic molecules.

van der Geest K.S., Kroesen B.J., Horst G., Abdulahad W.H., Brouwer E, & Boots A.M. (2018). Impact of Aging on the Frequency, Phenotype, and Function of CD161-Expressing T Cells. Frontiers in Immunology, 9, 752.

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Comprehensive Cytokine Profiling of Stimulated PBMC

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Blood samples (heparin) were diluted 1:1 with RPMI and stimulated with 40 nM PMA and 2 nM Ca²⁺ ionophore A23187 in the presence of 3 µM brefeldin A (all Sigma) for 4 h. Subsequently, red blood cells were lysed with ammonium chloride. The remaining cells were treated with Fix/Perm reagents A and B (Invitrogen) and stained with the following antibodies: CD3-eFluor 605, CD4-eFluor450, interleukin (IL)-17-AF488, Perforin-FITC (eBioscience), CD161-APC (Miltenyi), CD8-APC-H7, Granzyme-B-PE (BD), IFN-γ-PerCP-Cy5.5, IL-4-PE, and TNF-α-PerCP-Cy5.5 (Biolegend). Samples were measured on an LSR-II flow cytometer (BD) and analyzed with Kaluza Analysis Software (Beckman Coulter).

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Multiparametric Immune Cell Analysis

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CD3-PerCP-eFluor710, CD4-eFluor450, CD8-APC, NKp46-PE, FoxP3-FITC, CD69-FITC, rat IgG2a(κ)-FITC isotype control, armenian hamster IgG-FITC isotype control, rat IgG2a(κ)-PE isotype control, FoxP3 staining buffer set (eBioscience, USA). Gr1-FITC, CD11b-APC (Miltenyi Biotec, USA). Lin1-FITC (CD3, CD14, CD16, CD19, CD20, CD56), CD123-PE, HLA-DR-APC-H7, CD11c V450, CD69-PE, CD56-PE CD45-PerCP, human NK Cell Enrichment Set–DM, CD16-FITC, CD69-PE, CD4-PerCP (BD Biosciences, USA). CD19-FITC, CD11c-PerCP, MHC class II (I-A^d/I-E^d)-PE, CD80-FITC, CD86-APC, anti IL-12-PE, Rat IgG2a(κ)-APC Isotype Control (BD Pharmingen, USA), CD8a Pacific Blue, CD49b-APC (BioLegend, USA), CellTrace Violet™ Cell Proliferation kit (Invitrogen), CLI-095 (InvivoGen, USA). Pharmaceutical grade Immunomax® has been purchased from Immapharma Ltd (Russia).

Ghochikyan A., Pichugin A., Bagaev A., Davtyan A., Hovakimyan A., Tukhvatulin A., Davtyan H., Shcheblyakov D., Logunov D., Chulkina M., Savilova A., Trofimov D., Nelson E.L., Agadjanyan M.G, & Ataullakhanov R.I. (2014). Targeting TLR-4 with a novel pharmaceutical grade plant derived agonist, Immunomax®, as a therapeutic strategy for metastatic breast cancer. Journal of Translational Medicine, 12, 322.

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Whole Blood Immunophenotyping by Flow Cytometry

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Freshly drawn blood from an EDTA vacutainer was gently agitated to mix the contents, then 500 ul of whole blood was withdrawn and placed in a falcon tube. Seven millilitres of warmed (37 °C) BD lysis buffer (Cat No: 552052; BD, Oxford, UK) was added and the tube briefly vortexed to achieve red cell lysis. The mixture was diluted with phosphate-buffered saline (PBS) and centrifuged at 200 g for 10 minutes. The supernatant was discarded and the cellular pellet re-suspended in the residual fluid (approximately 200 ul). Fc blocker was added and the tube incubated in the dark for 10 minutes at room temperature (RT). Titrated volumes of colour-labelled antibodies (eBioscience, San Diego, CA, USA) were then added to the cell suspension: CD45 PerCP-Cy5.5 (45–0459), CD3 PE-Cy7 (25–0038), CD4 eFluor 450 (48–0047), CD8 APC-eFluor 780 (47–0088), CD56 APC (17–0567), δγ TCR FITC (11–9959), and CD69 PE (12–0699). The solution was incubated in the dark at RT for 15 minutes. The cells were washed with 2 ml PBS and centrifuged at 200 g for 5 minutes (×2). Then samples were fixed with 4 % paraformaldehyde and stored in the dark at 4 °C. Cytometer readings were performed within 48 h using a BD Canto II flow cytometer. Lymphocytes were identified using CD45 and side scatter.

Manson J., Cole E., De’Ath H.D., Vulliamy P., Meier U., Pennington D, & Brohi K. (2016). Early changes within the lymphocyte population are associated with the development of multiple organ dysfunction syndrome in trauma patients. Critical Care, 20, 176.

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