Ficoll density gradient centrifugation

Manufactured by PAN Biotech

Sourced in Germany

Ficoll density gradient centrifugation is a laboratory technique used for the separation and isolation of cells or other biological particles based on their density. It involves the use of a Ficoll solution, a synthetic polymer, to create a density gradient during centrifugation. This process allows for the selective separation of different cell types or components within a heterogeneous sample.

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6 protocols using ficoll density gradient centrifugation

Isolation and Stimulation of PBMCs

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Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (density: 1.077 g/ml; PanBiotech) and washed twice with DPBS (Gibco). Cells were resuspended in FBS (Gibco) containing 10% DMSO (Merck) and gradually frozen to -180 °C. For cell culture experiments, PBMCs were thawed, washed twice with DPBS, resuspended in B cell medium consisting of IMDM with L-Glutamine and 25 mM HEPES supplemented with 10% FBS, 1% Penicillin/Streptomycin, 1 mM Sodium Pyruvate, 1% MEM non-essential aminoacids (all from Gibco) and 0.055 mM β-Mercaptoethanol (Carl Roth) and seeded at a concentration of 0.25 × 10⁶ cells/well into 96-well round bottom plates. Cells were rested for 2 h at 37 °C and 5% CO₂ in a humified incubation chamber, if not differently described.
To analyze the effect of D₁-like receptor stimulation on cytokine release, 0.25 × 10⁶ PBMCs were seeded per well of a 96-well round bottom plate and stimulated with CpG ODN 2006 (0.35 μM, InvivoGen) with or without indicated concentrations of the agonists A68930 (Tocris) and SKF38393 (Tocris) for 24 h. Afterwards, cells were centrifuged and supernatants were frozen at − 80 °C until analysis.

Wieber K., Fleige L., Tsiami S., Reinders J., Braun J., Baraliakos X, & Capellino S. (2022). Dopamine receptor 1 expressing B cells exert a proinflammatory role in female patients with rheumatoid arthritis. Scientific Reports, 12, 5985.

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Isolation and Expansion of Human NK Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy donors by Ficoll density gradient centrifugation (PAN-Biotech, Germany). All blood donors gave their informed consent and all experiments were performed in accordance with relevant guidelines and regulations. Human NK cells were purified from PBMCs using the Dynabeads Untouched Human NK Cell kit (Thermo Fisher Scientific) according to manufacturer’s instructions. For NK cell activation and expansion, purified NK cells were cultured in 96-well round-bottom plates (Nunc) with irradiated K562-mbIL15-41BBL feeder cells (kind gift from Dario Campana) in IMDM Glutamax supplemented with 10% FCS and 1% penicillin/streptomycin, IL-2 (100 U/ml, NIH Cytokine Repository) and IL-15 (5 ng/ml, PAN-Biotech). Feeder cells were added at day 0 and day 7 of the culture and medium with fresh cytokines was exchanged every 2–3 days. IL-21 (100 ng/ml, Miltenyi Biotec) was added at the first day. NK cells were used for experiments between 3–6 weeks after isolation and they were between 90 and 99% CD3⁻, CD56⁺, and NKp46⁺ as assessed by flow cytometry.

Fasbender F, & Watzl C. (2018). Impedance-based analysis of Natural Killer cell stimulation. Scientific Reports, 8, 4938.

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Osteoclast Differentiation from PBMCs

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Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (density: 1.077 g/mL; PanBiotech, Aidenbach, Germany) and washed twice with DPBS (Gibco).
Cells were then resuspended in alphaMEM Glutamax (Gibco, Life technologies, Darmstadt, Germany) supplemented with 10% FCS, 100 U/mL penicillin (Life technologies, Darmstadt, Germany), 10 μg/mL streptomycin (Life technologies), and 25 ng/mL MCSF (BioLegend, Amsterdam The Netherlands). The cells were seeded at specific concentrations, depending on the culture plate used (1.5 × 10⁶ cells/well in 6 well plates, 0.15 × 10⁶ cells/well in 48 well plates, and 0.05 × 10⁶ cells/well in 96 well plates) and cultured at 37 °C, 5% CO₂. After 24 h, the medium was replaced with a differentiation medium containing alphaMEM Glutamax (Gibco, Life technologies, Darmstadt, Germany) supplemented with 10% FCS, 100 U/mL penicillin (Life technologies), 10 μg/mL streptomycin (Life technologies, Darmstadt, Germany), 25 ng/mL MCSF, and 50 ng/mL RANKL (BioLegend, Amsterdam The Netherlands). As an osteoclast differentiation control, cells were cultured without RANKL. The cells were cultured for 14 days, and the medium was replaced twice a week.

Schwendich E., Salinas Tejedor L., Schmitz G., Rickert M., Steinmeyer J., Rehart S., Tsiami S., Braun J., Baraliakos X., Reinders J., Neumann E., Müller-Ladner U, & Capellino S. (2022). Modulation of Dopamine Receptors on Osteoblasts as a Possible Therapeutic Strategy for Inducing Bone Formation in Arthritis. Cells, 11(10), 1609.

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Isolation of Primary CD14+ Monocytes

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Primary CD14+ monocytes were isolated from the peripheral blood by Ficoll density-gradient centrifugation (PAN-Biotech) and positive selection for CD14+ cells using MACS microbeads and the LS column system (Miltenyi Biotec) according to the manufacturer’s instructions.

Zierfuss B., Buda A., Villoria-González A., Logist M., Fabjan J., Parzer P., Battin C., Vandersteene S., Dijkstra I.M., Waidhofer-Söllner P., Grabmeier-Pfistershammer K., Steinberger P., Kemp S., Forss-Petter S., Berger J, & Weinhofer I. (2022). Saturated very long-chain fatty acids regulate macrophage plasticity and invasiveness. Journal of Neuroinflammation, 19, 305.

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Isolation and Activation of Human NK Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors by Ficoll density gradient centrifugation (PAN-Biotech, Aidenbach Germany). Human NK cells were purified from PBMCs using the Dynabeads Untouched Human NK Cell kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to manufacturer's instructions. For NK cell activation and expansion, purified NK cells were cultured in 96-well round-bottom plates (Nunc) with irradiated K562-mbIL15-41BBL (kind gift from Dario Campana) in IMDM Glutamax supplemented with 10% FCS and 1% penicillin/streptomycin, IL-2 (100 U/ ml, NIH Cytokine Repository) and IL-15 (5 ng/ml, PAN-Biotech). IL-21 (100 ng/ml, Miltenyi Biotec, Bergisch Gladbach Germany) was added at the first day. NK cells were between 90 and 99% CD3 -, CD56 + , and NKp46 + as assessed by flow cytometry. Huh7 cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) containing 4.5% glucose, 1% penicillin/streptomycin mixture and 10% heat-inactivated FCS. Primary human hepatocytes were cultivated in William's E medium (PAN Biotech, P04_29510) with 100 U/ml penicillin, 0,1 mg/ml streptomycin, 10 μg/ml gentamicin, 2 mM stable glutamine, 100 nM dexamethasone and 2 nM insulin-transferrin-selenite (ITS) supplement.
When plating cells, 10% fetal calf serum was added for the first 3-4 h of cultivation.

Fasbender F., Obholzer M., Metzler S., Stöber R., Hengstler J.G, & Watzl C. (2020). Enhanced activation of human NK cells by drug-exposed hepatocytes. Archives of toxicology, 94(2).

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Isolation and Culture of Primary and EBV-Immortalized B Cells

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Primary B cells were isolated from peripheral blood by Ficoll density-gradient centrifugation (PAN-Biotech) and positive selection for CD19+ cells using MACS microbeads and the LS column system (Miltenyi Biotec) according to the manufacturer’s instructions. The purity of isolated primary B cells was determined using a mouse anti-human CD19 monoclonal antibody, PE conjugated (clone LT19, # 130-091-247, Miltenyi) and the PE-conjugated monoclonal antibody isotype control (#120-002-723, Miltenyi) by flow cytometry. Primary B cells isolated from the blood and EBV-immortalized B lymphocytes kindly provided by Dr. Ann Moser, Kennedy Krieger Institute, Baltimore, USA and Prof. Florian Eichler, Harvard Medical School, Boston, USA, were maintained in RPMI complete medium [RPMI 1640 supplemented with 2 mM L-glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin (all Invitrogen) and 10% heat-inactivated fetal calf serum (Gibco Life Technologies)]. EBV-immortalized B lymphocytes were treated with 2 µM 25-HC or the solvent ethanol for the indicated time period. HEK-293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) supplemented as described above for RPMI. All cells were cultivated at 37 °C and 5% CO₂.

Weinhofer I., Buda A., Kunze M., Palfi Z., Traunfellner M., Hesse S., Villoria-Gonzalez A., Hofmann J., Hametner S., Regelsberger G., Moser A.B., Eichler F., Kemp S., Bauer J., Kühl J.S., Forss-Petter S, & Berger J. (2022). Peroxisomal very long-chain fatty acid transport is targeted by herpesviruses and the antiviral host response. Communications Biology, 5, 944.

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