Ficoll density separation

Manufactured by GE Healthcare

Ficoll density separation is a laboratory technique used to isolate specific types of cells or other biological particles from a complex mixture. It utilizes a density gradient medium composed of sucrose and Ficoll polymer to facilitate the separation of different cell types based on their density differences. The core function of this method is to allow for the efficient and gentle isolation of cell populations, such as mononuclear cells, for further analysis or downstream applications.

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Lab products found in correlation

Cd3 fitc, by BioLegend (2 mentions) Cd56 apc, by BioLegend (2 mentions) Facscanto 2, by BD (2 mentions) Cd8 apc, by BioLegend (1 mentions) Cd4 pe, by BioLegend (1 mentions) Cd45ra pe, by BioLegend (1 mentions) Cd45ro apc, by BioLegend (1 mentions) Anti cd3 plus anti cd28 antibodies, by BioLegend (1 mentions) Phytohemagglutinin (pha), by Merck Group (1 mentions) Cytofix cytoperm kit, by BD (1 mentions)

2 protocols using ficoll density separation

Evaluation of Lymphocyte Subsets and Proliferation

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Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density separation (GE healthcare, Life Systems). For the evaluation of different lymphocyte subsets, PBMCs were stained with the following fluorochrome-conjugated monoclonal antibodies against the following cell surface markers: CD3 FITC (Biolegend, clone OKT3), CD4 PE (Biolegend, clone OKT4), CD8 APC (Biolegend, clone SK1), CD19 PerCP-Cy5 (Biolegend, clone 4G7), CD56 APC (Biolegend, clone 5.1H11), CD45 RA PE (Biolegend, clone HI100), and CD45 RO APC (Biolegend, clone UCHL1). For proliferation assays, PBMCs were stained with CellTracker™ green CMDFA dye (ThermoFisher Scientific, USA). Cells were stimulated with purified anti-CD3 plus anti-CD28 antibodies (BioLegend, USA) or PHA (Sigma) for 5 days at 37 °C 5% CO₂. Before sample acquisition, PBMCs were stained using the murine PE-conjugated monoclonal antibody against human CD3 (Biolegend, clone OKT3). All samples were acquired on FACSCanto II (BD bioscience) and analyzed with the use of FlowJo 7.6.5 software (Tree Star, Ashland, OR).

Lugo-Reyes S.O., Pastor N., González-Serrano E., Yamazaki-Nakashimada M.A., Scheffler-Mendoza S., Berron-Ruiz L., Wakida G., Nuñez-Nuñez M.E., Macias-Robles A.P., Staines-Boone A.T., Venegas-Montoya E., Alaez-Verson C., Molina-Garay C., Flores-Lagunes L.L., Carrillo-Sanchez K., Niemela J., Rosenzweig S.D., Gaytan P., Yañez J.A., Martinez-Duncker I., Notarangelo L.D., Espinosa-Padilla S, & Cruz-Munoz M.E. (2021). Clinical Manifestations, Mutational Analysis, and Immunological Phenotype in Patients with RAG1/2 Mutations: First Cases Series from Mexico and Description of Two Novel Mutations. Journal of clinical immunology, 41(6), 1291-1302.

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Evaluating SAP Expression in NK Cells

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Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll density separation (GE healthcare, Life Systems). For the evaluation of intracellular expression of SAP in NK cells, the PBMCs were stained with the following panel of fluorochrome-conjugated monoclonal Abs directed against cell surface markers: CD3 FITC (Biolegend, clone OKT3), CD56 APC (Biolegend, clone 5.1H11). Then, cells were fixed and permeabilized using Cytofix/Cytoperm Kit (BD Bioscience) and finally stained using the murine PE conjugated monoclonal antibody directed against human SAP (Thermo Scietific, clone XLP 1D12). An isotype control was used for every staining. Flow cytometric data were acquired on FACSCanto II (BD bioscience) and analyses with the use of FlowJo 7.6.5 software (Tree Star, Ashland, OR). Gates were set to exclude CD3⁺lymphocytes. Thereafter, NK cells were defined by the expression of CD56. The MFI SAP expression in NK cells was determined by using isotype control. SAP expression was analyzed in 18 B-ALL patients and 14 age-matched healthy controls.

Valenzuela-Vazquez L., Núñez-Enríquez J.C., Sánchez-Herrera J., Jiménez-Hernández E., Martín-Trejo J.A., Espinoza-Hernández L.E., Medina-Sanson A., Flores-Villegas L.V., Peñaloza-González J.G., Refugio Torres-Nava J., Espinosa-Elizondo R.M., Amador-Sánchez R., Santillán-Juárez J.D., Flores-Lujano J., Pérez-Saldívar M.L., García-López L.R., Castañeda-Echevarría A., Rodríguez-Leyva F., Rosas-Vargas H., Mata-Rocha M., Duarte-Rodríguez D.A., Sepúlveda-Robles O.A., Mancilla-Herrera I., Mejía-Aranguré J.M, & Cruz-Munoz M.E. (2020). Functional characterization of NK cells in Mexican pediatric patients with acute lymphoblastic leukemia: Report from the Mexican Interinstitutional Group for the Identification of the Causes of Childhood Leukemia. PLoS ONE, 15(1), e0227314.

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