Ficoll paque

Manufactured by Corning

Sourced in United States

Ficoll-Paque is a density gradient medium used for the separation and isolation of cells, such as lymphocytes, from whole blood or other biological samples. It is a sterile, pyrogen-free, and isotonic solution that enables the efficient separation of different cell types based on their density differences.

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Lab products found in correlation

Lymphocyte separation medium, by Corning (3 mentions) Dimethyl sulfoxide (dmso), by Merck Group (3 mentions) Prescreened fbs, by Gemini Bio (3 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Magnetic activated cell sorter, by Miltenyi Biotec (1 mentions) Nk cell isolation kit, by Miltenyi Biotec (1 mentions) Comirnaty, by Pfizer (1 mentions) Bnt162b2, by Pfizer (1 mentions) Human ab serum, by Corning (1 mentions) Ficoll paque, by GE Healthcare (1 mentions) Streptomycin, by Corning (1 mentions) Dulbecco modified eagle medium (dmem), by Corning (1 mentions) L glutamine, by Corning (1 mentions) Rpmi 1640, by Corning (1 mentions) Sars cov 2 igg 2 quant, by Abbott (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Recombinant il 2, by Thermo Fisher Scientific (1 mentions) Phytohemagglutinin (pha), by Thermo Fisher Scientific (1 mentions)

8 protocols using ficoll paque

PBMC Isolation and NK Cell Purification for COVID-19 Research

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Using whole blood samples obtained from patients with COVID-19, patients with influenza A, and healthy donors, we isolated PBMCs by Ficoll-Paque density gradient centrifugation using lymphocyte separation medium (Corning, New York, NY). PBMCs were cryopreserved until use.
After thawing cryopreserved PBMCs, the cell viability was 86.88% ± 7.35% (healthy donors), 69.21% ± 13.77% (patients with influenza), 79.75% ± 12.11% (patients with mild COVID-19), and 75.22% ± 10.19% (patients with severe COVID-19) (see Table E5 in this article’s Online Repository at www.jacionline.org). Thawed PBMCs were directly analyzed without in vitro incubation.
NK cells were isolated 2 ways. For RNA-seq, we stained PBMCs with antibodies against 7-AAD, CD3, and CD56, and sorted NK cells on the basis of surface expression of CD3 and CD56 (CD3^–CD56⁺) using an ARIA II cell sorter. The gating strategies for NK-cell sorting are presented in Fig E1 in this article’s Online Repository at www.jacionline.org. For the NK-cell cytotoxicity assay, to avoid affecting NK cells as much as possible, we negatively isolated NK cells using the NK-cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). We stained other cells except NK cells in the PBMCs according to the manufacturer’s instructions and obtained NK cells by removing magnetically labeled cells using a magnetic-activated cell sorter (Miltenyi Biotec).

Leem G., Cheon S., Lee H., Choi S.J., Jeong S., Kim E.S., Jeong H.W., Jeong H., Park S.H., Kim Y.S, & Shin E.C. (2021). Abnormality in the NK-cell population is prolonged in severe COVID-19 patients. The Journal of Allergy and Clinical Immunology, 148(4), 996-1006.e18.

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Longitudinal Study of COVID-19 Vaccine Responses

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This longitudinal study included 79 Caucasian health care workers followed since March 2020 in the tertiary Ramon y Cajal University Hospital (Madrid, Spain) who were vaccinated with two doses of Comirnaty (mRNA-based BNT162b2, Pfizer-BioNTech) COVID-19 vaccine in February 2021. Twenty-four individuals had specific antibodies and 55 individuals were seronegative. The complete study design is shown in Figure 1A.
Peripheral blood mononuclear cells (PBMC) were isolated from EDTA-blood sample by Ficoll-Paque density gradient centrifugation using lymphocyte separation medium (Corning, New York, NY) and cryopreserved. Plasma samples were stored at −80°C.
All individuals included in the study provided either oral or written informed consent. This study was conducted in accordance with the Declaration of Helsinki (1996), approved by the institutional review boards of our Hospital Ethics Committee (EC162/20), and registered at the clinical trials repository.¹ Demographic data including age, sex, race, body mass index (BMI), and comorbidities were recorded for all participants.

Vallejo A., Vizcarra P., Martín-Hondarza A., Gómez-Maldonado S., Haemmerle J., Velasco H, & Casado J.L. (2022). Impact of SARS-CoV-2-specific memory B cells on the immune response after mRNA-based Comirnaty vaccine in seronegative health care workers. Frontiers in Microbiology, 13, 1002748.

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Isolation and Differentiation of Immune Cells

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Human leukocytes were isolated from whole blood of healthy donors as buffy coats following Ficoll-Paque^™ (GE Healthcare, 17-1440-02) density gradient centrifugation. Cells were washed three times in phosphate buffered saline (PBS) (Corning, 21-040-CV) to remove residual Ficoll-Paque^™. Adherent monocytes were isolated from lymphocytes and differentiated into macrophages over ten days in Dulbecco’s Modified Eagle Medium (DMEM) (Corning, #13619003) supplemented with 10% human AB serum (Corning, # 35060CI).
HEK293T (ATCC) and HeLa (ATCC) cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Sigma, #F4135), 100 I.U. pencillin (per mL), 100 μg/mL streptomycin and 292 μg/mL L-glutamine (100X stock, Corning, #30-009-CI). SupT1 (ATCC), THP-1 (ATCC) and SAMHD1 KO THP-1 (a gift from Baek Kim) cell lines were cultured in Roswell Park Memorial Institute 1640 media (RPMI-1640) (Corning, #10-040-CV) supplemented with 10% fetal bovine serum (FBS), 100 I.U. penicillin (per mL), 100 μg/mL streptomycin and 292 μg/mL L-glutamine. Where noted, THP-1 cells were differentiated into a macrophage-like state by treatment with 120 ng/mL phorbol myristate acetate (PMA) (Fisher, #16561-29-8) for 2 days before described treatments. Treatments were applied in the presence of PMA. All cells were cultured at 37°C in a humidified chamber with 5% CO2.

Rabinowitz J., Sharifi H.J., Martin H., Marchese A., Robek M., Shi B., Mongin A.A, & de Noronha C.M. (2021). xCT/SLC7A11 Antiporter Function Inhibits HIV-1 Infection. Virology, 556, 149-160.

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Serological Profiling of COVID-19 Vaccines

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Peripheral blood mononuclear cells (PBMC) were isolated from EDTA blood samples by Ficoll-Paque density gradient centrifugation using lymphocyte separation medium (Corning, New York, NY) and cryopreserved. Plasma samples were stored at -80°C.
All the participants (naïve and convalescent) were tested for anti-N SARS-CoV-2 IgG antibodies (COVID-19-SARS-CoV-2 IgG ELISA, Demeditech, Germany) at inclusion and after a median of 17 days after the second vaccine dose to confirm serological status independent of the antibody production to the vaccine. Results were expressed as relative units per milliliter (U/mL), with a cut-off of 11 U/mL. They were also tested for anti-Spike IgG antibodies (SARS-CoV-2 IgG II Quant, Abbott, Maidenhead, United Kingdom) with a cut-off of 50 arbitrary units per milliliter (AU/mL) and for both anti-S IgA and anti-S IgM (COVID-19-SARS-CoV-2 IgA ELISA, COVID-19-SARS-CoV-2 IgM ELISA Demeditech, Germany), with a threshold of 0.1 U/mL at baseline and after both doses. All these assays used the SARS-CoV-2 B.1.351 lineage (beta variant) for antibody detection.

Casado J.L., Vizcarra P., Martín-Hondarza A., Gómez-Maldonado S., Muedra-Sánchez M., del Pino J., Mirabella I.G., Martín-Colmenarejo S., Haemmerle J., Fernández-Escribano M, & Vallejo A. (2023). mRNA-based SARS-CoV-2 Comirnaty vaccine elicits weak and short specific memory B cell response in individuals with no previous infection. Frontiers in Immunology, 14, 1127379.

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Culturing Cell Lines and PBMCs

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The Raji cell line (Burkitt’s lymphoma) and Jurkat cell line (acute T-lymphoblastic leukemia) were kindly provided by Assistant Professor Dachit Nilubol and Professor Kovit Pattanapanyasat. Both cell lines were grown in RPMI-1640 medium (Hyclone, USA) with 10% FBS (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA).
Human peripheral mononuclear cells (PBMCs), obtained from healthy donors, were isolated by density gradient centrifugation using Ficoll-Paque (Corning, USA). The PBMCs were then cultured in RPMI 1640 medium with 10% fetal bovine serum and 1% penicillin/streptomycin.
For the generation of allogeneic cells, PBMCs were stimulated with 5 μg/ml phytohemagglutinin (PHA) (Invitrogen, USA.) in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin and 100 U/ml recombinant IL-2 (PeproTech, USA). The allogeneic cells were cultured for seven days, during which the medium and IL-2 were replaced. The medium and IL-2 of viable cells were changed every 2–3 days. All cells were maintained at 37 °C with 5% CO₂. The study was approved by the Ethical Clearance Committee on Human Rights Related to Research Involving Human Subjects, Faculty of Medicine Ramathibodi Hospital, Mahidol University (MURA2020/879). The written consents were obtained from all the participants involved in this study.

Sawaisorn P., Atjanasuppat K., Uaesoontrachoon K., Rattananon P., Treesuppharat W., Hongeng S, & Anurathapan U. (2023). Comparison of the efficacy of second and third generation lentiviral vector transduced CAR CD19 T cells for use in the treatment of acute lymphoblastic leukemia both in vitro and in vivo models. PLOS ONE, 18(2), e0281735.

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Isolation of Mononuclear Cells from Cord Blood

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All processing was completed using BSL 2 procedures. Prior to cell isolation, a cord blood aliquot was diluted with one part 10% RPMI to one part whole blood in a sterile 50 mL conical tube. Diluted blood was layered over Ficoll-Paque® (Lymphocyte Separation Medium, Cellgro) at a ratio of 3-mL blood to 1-mL separation medium. Blood was pipetted slowly down the side of the 50 mL conical to overlay the Ficoll-Paque. Samples were centrifuged at 20°C for 30 min at 900g with no brake. After centrifugation the mononuclear cell layer was removed and transferred to a new 50mL conical. The tube was filled with 5% RPMI and centrifuged for 5 minutes at 500g at 20°C, with the brake. Cells were washed with 5% RPMI, and then resuspended in 10% RPMI. Cells were frozen in 10×10⁶ cells/mL aliquots in freeze media, 10% DMSO (Sigma) and 90% sterile-filtered prescreened FBS (Gemini Bioproducts). Cells were kept in long term storage in −150 degree freezers.

Fike A.J., Nguyen L.T., Kumova O.K, & Carey A.J. (2017). Characterization of CD31 expression on murine and human neonatal T lymphocytes during development and activation. Pediatric research, 82(1), 133-140.

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Ficoll Isolation of Mononuclear Cells

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All processing was completed using BSL 2 procedures. Prior to cell isolation, a blood aliquot was diluted with one part 10% RPMI to one part whole blood in a sterile 50 mL conical tube. Diluted blood was layered over Ficoll-Paque^® (Lymphocyte Separation Medium, Cellgro) at a ratio of 3 mL blood to 1 mL separation medium. Blood was pipetted slowly down the side of the 50 mL conical to overlay the Ficoll-Paque. Samples were centrifuged at 20°C for 30 min at 900 g with no brake. After centrifugation the mononuclear cell layer was removed and transferred to a new 50 mL conical. The tube was filled with 5% RPMI and centrifuged for 5 min at 500 g at 20°C, with the brake. Cells were washed with 5% RPMI, and then resuspended in 10% RPMI. Cells were frozen in 1 × 10⁷ cells/mL aliquots in freeze media, 10% DMSO (Sigma) and 90% sterile-filtered prescreened FBS (Gemini Bioproducts). Cells were kept in long-term storage in −150° freezers.

Carey A.J., Hope J.L., Mueller Y.M., Fike A.J., Kumova O.K., van Zessen D.B., Steegers E.A., van der Burg M, & Katsikis P.D. (2017). Public Clonotypes and Convergent Recombination Characterize the Naïve CD8+ T-Cell Receptor Repertoire of Extremely Preterm Neonates. Frontiers in Immunology, 8, 1859.

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Isolation of Mononuclear Cells from Cord Blood

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