Ficoll paque lymphoprep

Manufactured by STEMCELL

Sourced in France

Ficoll-Paque is a sterile, pyrogen-tested density gradient medium for the isolation of mononuclear cells from blood or bone marrow samples. It is composed of sucrose and sodium diatrizoate, with a density of 1.077 g/mL. When used for cell separation, Ficoll-Paque allows the selective isolation of mononuclear cells, such as lymphocytes and monocytes, from whole blood or bone marrow.

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5 protocols using ficoll paque lymphoprep

COVID-19 Serosurvey and PBMC Isolation

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The study was performed in accordance with the Declaration of Helsinki and reviewed for approval by the Ethics Committee of Tiantan Biological R&D Center (approval No. TTSW-EC2021-05). All participants were over 18 years old, and written informed consent was obtained at the time of their enrollment. All participants had no breakthrough infection or reinfection during the period between primary infection and blood collection.
Serum (10 mL of whole blood) or PBMCs (15 mL of whole blood) were collected into a blood collection tube (Chengwu Medical, China) containing procoagulant or heparin sodium. Serum samples were obtained immediately after centrifugation at 1500 rpm for 15 min and then stored at −20 °C for serology assays. PBMCs were obtained via density gradient centrifugation using Ficoll-Paque (Lymphoprep, STEMCELL Technologies) according to the guidance for use. The isolated PBMCs were resuspended in cryopreserved cell recovery medium containing 10% dimethyl sulfoxide (DMSO; Gibco) and 90% heat-inactivated fetal bovine serum (Gibco) and then stored in liquid nitrogen until cellular assays were conducted.

Liang H., Nian X., Wu J., Liu D., Feng L., Lu J., Peng Y., Zhou Z., Deng T., Liu J., Ji D., Qiu R., Lin L., Zeng Y., Xia F., Hu Y., Li T., Duan K., Li X., Wang Z., Zhang Y., Zhang H., Zhu C., Wang S., Wu X., Wang X., Li Y., Huang S., Mao M., Guo H., Yang Y., Jia R., Xufang J., Wang X., Liang S., Qiu Z., Zhang J., Ding Y., Li C., Zhang J., Fu D., He Y., Zhou D., Li C., Zhang J., Yu D, & Yang X.M. (2022). COVID-19 vaccination boosts the potency and breadth of the immune response against SARS-CoV-2 among recovered patients in Wuhan. Cell Discovery, 8, 131.

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PBMC Isolation and Characterization

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PBMCs were isolated by differential centrifugation over Ficoll-Paque (Lymphoprep, StemCell Technologies, Inc.). Cells were washed three times in PBS. PBMCs and monocytes were resuspended in RPMI culture medium supplemented with 2mM glutamax, 1mM pyruvate and penicillin/streptomycin (all from Thermo Fisher Scientific) and counted on a Casy counter. Cell counts of whole blood and isolated PBMCs were also analyzed using a sysmex XN-450 automated hematology analyzer (Sysmex).

Rother N., Yanginlar C., Lindeboom R.G., Bekkering S., van Leent M.M., Buijsers B., Jonkman I., de Graaf M., Baltissen M., Lamers L.A., Riksen N.P., Fayad Z.A., Mulder W.J., Hilbrands L.B., Joosten L.A., Netea M.G., Vermeulen M., van der Vlag J, & Duivenvoorden R. (2020). Hydroxychloroquine Inhibits the Trained Innate Immune Response to Interferons. Cell Reports Medicine, 1(9), 100146.

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Differentiation of Monocytes to Dendritic Cells

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Human Peripheral Blood Mononuclear Cells were prepared from buffy coats (Etablissement Francais du Sang (EFS), Lille, France) using Ficoll Paque (Lymphoprep, StemCell). The use of human samples was approved by the French Ministry of Education and Research under the agreement DC 2013-2575. According to French Public Health Law (art L 1121–1-1, art L 1121–1-2), Institutional Review Board and written consent approval are not required for human non-interventional studies. Monocytes were positively isolated using CD14⁺ microbeads (Miltenyi Biotec) according to the manufacturer’s recommendations. Cells were cultured for 6 days with rh GM-CSF (20ng/ml; Peprotech) and rh IL-4 (5ng/ml; Peprotech). When mentioned, muramyl dipeptide (MDP) (10μg/ml; Invitrogen) was added from the beginning of a 5-day culture with GM-CSF and IL-4 (day 0). The mTOR activator MYH1485 (2μM, Sigma), wortmannin (1μM, Sigma) or rapamycin (100nM, Sigma) were added at the start of the culture (day 0). Adalimumab (Humira M02‐497) was a gift from Abbott (Abbott Park, IL, USA).

Chauvin C., Alvarez-Simon D., Radulovic K., Boulard O., Laine W., Delacre M., Waldschmitt N., Segura E., Kluza J., Chamaillard M, & Poulin L.F. (2023). NOD2 in monocytes negatively regulates macrophage development through TNFalpha. Frontiers in Immunology, 14, 1181823.

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In vitro study of glomerular endothelial and immune cell responses

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Mouse glomerular endothelial cells (mGEnC-1) were cultured as described previously (Rops et al., 2004b (link)). Differentiated mGEnC-1 were stimulated with 100 ng/mL LPS (O111:B4; Sigma-Aldrich) and treated with 10 μg/mL of unstimulated HS_glx, LPS HS_glx, Fucoidan or sulodexide. Treatment was added 1 h prior to LPS stimulation.
Human peripheral blood mononuclear cells (PBMCs) were isolated by differential centrifugation over Ficoll-Paque (Lymphoprep, StemCell Technologies, Inc.). PBMCs were washed three times in PBS, resuspended in RPMI culture medium supplemented with 2 mM glutamax, 1 mM pyruvate and penicillin/streptomycin (all from Thermo Fisher Scientific, Breda, Netherlands), and counted on a Casy counter. PBMCs from at least three different donors were seeded into 96-well flat bottom plates in a density of 500,000 cells per well. Cells were allowed to adhere for 1 h at 37°C. Cells were washed three times with PBS prior to stimulations. PBMCs were stimulated with 10 ng/mL LPS (E. coli 055:B5; Cat#trlrl-pb5LPS; Invivogen, Huissen, Netherlands) and 10 μg/mL of unstimulated HS_glx, LPS HS_glx, fucoidan or sulodexide. Treatment was added 1 h prior to LPS stimulation.

Buijsers B., Maciej-Hulme M., Jacobs M., Bebber M.B., de Graaf M., Salmenov R., Parr N., Rabelink T.J., Nijenhuis T, & van der Vlag J. (2023). Glycosaminoglycans and fucoidan have a protective effect on experimental glomerulonephritis. Frontiers in Molecular Biosciences, 10, 1223972.

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Isolation and Quantification of PBMCs

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PBMCs were isolated by differential centrifugation over Ficoll-Paque (Lymphoprep, Stemcell Technologies). Cells were washed three times in PBS. PBMCs and monocytes were resuspended in RPMI culture medium supplemented with 2mM glutamax, 1mM pyruvate and penicillin/streptomycin (all from Thermo Fisher Scientific) and counted on a Casy counter. Cell counts of whole blood and isolated PBMCs were also analyzed using a sysmex XN-450 automated hematology analyzer (Sysmex).

Rother N., Yanginlar C., Lindeboom R.G., Bekkering S., Leent M.M.T.v., Buijsers B., Jonkman I., Graaf M.d., Baltissen M.P., Lamers L.A., Deitch E.A., Fayad Z.A., Mulder W.J.M., Hilbrands L.B., Joosten L.A.B., Netea M.G., Vermeulen M., Vlag J.v.d., & Duivenvoorden R. (2020). Hydroxychloroquine inhibits trained immunity – implications for COVID-19.

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