Ficoll separation

Manufactured by GE Healthcare

Sourced in Sweden, United States

Ficoll separation is a laboratory technique used to isolate and purify specific cell populations from a heterogenous mixture, such as blood or bone marrow. It utilizes a density gradient medium called Ficoll to separate cells based on their density differences. The core function of Ficoll separation is to enable the isolation of mononuclear cells, such as lymphocytes and monocytes, from other blood components in a gentle and efficient manner.

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Lab products found in correlation

Anti cd11b microbead, by Miltenyi Biotec (2 mentions) Anti fluorescein isothiocyanate microbeads, by Miltenyi Biotec (2 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Huvecs, by Lonza (1 mentions) Dnase, by Worthington (1 mentions) Hek293, by American Type Culture Collection (1 mentions) Human umbilical vein endothelial cells (huvec), by American Type Culture Collection (1 mentions) Mcf 7, by American Type Culture Collection (1 mentions) Caco 2, by American Type Culture Collection (1 mentions) Anti nk1, by BioLegend (1 mentions) Anti cd19, by BioLegend (1 mentions) Anti ly6g, by BioLegend (1 mentions) Macs column, by Miltenyi Biotec (1 mentions) Anti fitc microbeads, by Miltenyi Biotec (1 mentions) Ifn γ elispot plates, by R&D Systems (1 mentions) Human gm csf, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Cellstripper, by Corning (1 mentions) Ultra low attachment 6 well plate, by Corning (1 mentions)

Close spelling variants of this product

Ficoll-Paque Plus separation medium Lymphocyte separation medium Ficoll Ficoll-Paque gradient separation Ficoll density separation Ficoll-Paque separation media Ficoll-Paque separation medium Ficoll gradient separation Ficoll–Hypaque gradient separation Ficoll-paque PLUS gradient separation Human Lymphocyte separation medium Ficoll

12 protocols using ficoll separation

Isolation and Culture of Human Endothelial Cells

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Human umbilical vein endothelial cells (HUVECs; Lonza, Walkersville, MD; C2517A) were cultured using EGM-2 MV media (Lonza; CC-3202). KG-1 human leukemia cells (ATCC, Manassas, VA; CCL-246) were grown in IMDM (Hyclone, Fisher Scientific, Hanover Park, IL; SH30228.01) plus 20% FBS.
Bone marrow endothelial cells (BMECs) were freshly isolated from healthy human bone marrow (Lonza). Briefly, mononuclear cells were isolated from fresh bone marrow samples using Ficoll separation (GE Healthcare, Chicago, IL; GE17-1440-02) and plated in collagen-coated plates. Adherent cells were collected and cultured using EGM-2 MV media supplemented with 10% FBS to generate the BMECs.

Vijay V., Miller R., Vue G.S., Pezeshkian M.B., Maywood M., Ast A.M., Drusbosky L.M., Pompeu Y., Salgado A.D., Lipten S.D., Geddes T., Blenc A.M., Ge Y., Ostrov D.A., Cogle C.R, & Madlambayan G.J. (2019). Interleukin-8 Blockade Prevents Activated Endothelial Cell Mediated Proliferation and Chemoresistance of Acute Myeloid Leukemia. Leukemia research, 84, 106180.

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Isolation and Cryopreservation of Normal and AML Cells

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Normal donor human blood cells were obtained fresh from AllCells (Alameda, CA) and FACS-enriched. Human AML samples were obtained from patients at the Stanford Medical Center with informed consent, according to Institutional Review Board (IRB)-approved protocols (Stanford IRB no 18329 and 6453). Mononuclear cells were isolated from each AML sample via Ficoll separation (GE Healthcare Life Sciences, Pittsburg, PA) and cryopreserved in liquid nitrogen in 90% FBS + 10% DMSO. All experiments presented here were conducted on freshly thawed cells. Criteria for inclusion of AML samples in this study were pre-established. Samples were included based on blast percentage at sample collection. For normal donors, no exclusion criteria were used. All AML samples were thawed in IMDM + 10% FBS + 200 U/ml DNase (Worthington Biochemical, Lakewood, NJ)

McKeown M.R., Corces M.R., Eaton M.L., Fiore C., Lee E., Lopez J.T., Chen M.W., Smith D., Chan S.M., Koenig J.L., Austgen K., Guenther M.G., Orlando D.A., Lovén J., Fritz C.C, & Majeti R. (2017). Super-Enhancer analysis defines novel epigenomic subtypes of non-APL AML including an RARα dependency targetable by SY-1425, a potent and selective RARα agonist. Cancer discovery, 7(10), 1136-1153.

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Isolation and Culture of Various Cell Types

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Chinese hamster ovary cells (CHO), human embryonic kidney cells (HEK 293), human umbilical vein endothelial cells (HUVEC) and human epithelial colorectal adenocarcinoma cells (CACO-2) human breast cancer epithelial cells (MCF-7), in addition to primary human cells were purchased from ATCC. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples obtained from healthy volunteers using Ficoll separation (GE Healthcare) as described by the manufacturer. Healthy volunteer blood samples were procured with signed informed consent and IRB approval by Western IRB. Patient blood samples were collected with signed informed consent and an IRB with Inova Fairfax Hospital.

Stefansson S., Adams D.L., Ershler W.B., Le H, & Ho D.H. (2016). A cell transportation solution that preserves live circulating tumor cells in patient blood samples. BMC Cancer, 16, 300.

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Isolating PBMCs and Spleen Cells

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Peripheral Blood Mononuclear Cells (PBMCs) were isolated from heparinized blood by use of standard Ficoll separation (GE Healthcare, Uppsala, Sweden) and stored at −140°C until use. Donor spleen cells were obtained by mechanical dissociation of spleen from the organ donor. After filtration of the cell suspension, the cells were isolated by Ficoll separation.

Mendoza Rojas A., Verhoeven J.G., de Kuiper R., Clahsen-van Groningen M.C., Boer K., Hesselink D.A., van Gelder T., van Besouw N.M, & Baan C.C. (2023). Alloreactive T cells to Assess Acute Rejection Risk in Kidney Transplant Recipients. Transplantation Direct, 9(5), e1478.

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Isolation of Myeloid and Lymphoid Cells

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Wounds were digested as described above. Single cell suspensions were incubated with fluorescein isothiocyanate–labeled anti-CD3, anti-CD19, and anti-Ly6G (BioLegend) followed by anti–fluorescein isothiocyanate microbeads (Miltenyi Biotec). Flow-through was then incubated with anti-CD11b microbeads (Miltenyi Biotec) to isolate the non-neutrophil, non-lymphocyte, CD11b+ cells. Cells were saved in Trizol (Invitrogen) for quantitative RT-PCR analyses. For human monocyte isolation, peripheral blood was collected and subjected to RBC lysis and Ficoll separation (GE healthcare). Cell suspensions were then treated with anti-human CD14 microbeads. Magnetic separation yielded 95% purity by flow cytometry.

Davis F.M., denDekker A., Kimball A., Joshi A.D., Azzouny M.E., Wolf S.J., Obi A.T., Lipinski J., Gudjonsson J.E., Xing X., Plazyo O., Audu C., Melvin W.J., Singer K., Henke P.K., Moore B.B., Burant C., Kunkel S.L, & Gallagher K.A. (2020). Epigenetic Regulation of TLR4 in Diabetic Macrophages Modulates Immunometabolism and Wound Repair. Journal of immunology (Baltimore, Md. : 1950), 204(9), 2503-2513.

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Synovial Fluid and PBMC Analysis in ACPA+ and ACPA- RA

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This study was approved by the research ethics committee of Karolinska University Hospital, Sweden and all patients signed informed consent according to the Declaration of Helsinki. All patients had established disease, based on the 1987 criteria from the American College of Rheumatology. Synovial fluid from ACPA− (n = 13) and ACPA+ (n = 16) RA patients were collected at several visits at the Rheumatology Clinic of Karolinska University Hospital, Stockholm (Supplementary Data 1). In n = 12 ACPA− and n = 10 ACPA+ RA patients, paired PBMCs were also available. PBMC and SFMC were isolated by Ficoll separation (GE Healthcare) and cryopreserved in fetal bovine serum (FBS) with 10% DMSO in liquid nitrogen.

Argyriou A., Wadsworth MH I.I., Lendvai A., Christensen S.M., Hensvold A.H., Gerstner C., van Vollenhoven A., Kravarik K., Winkler A., Malmström V, & Chemin K. (2022). Single cell sequencing identifies clonally expanded synovial CD4+ TPH cells expressing GPR56 in rheumatoid arthritis. Nature Communications, 13, 4046.

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Longitudinal Profiling of Rheumatoid Arthritis

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Peripheral blood and synovial tissue samples were collected before treatment (baseline (FU0)), at week 4 (follow-up 1 (FU1)) and at week 16 (follow-up 2 (FU2)) after rituximab treatment, as described before.12 14 (link) For some patients the week 16 post-treatment peripheral blood sample was not available and peripheral blood samples collected at week 24 were analysed instead. Peripheral blood mononuclear cells (PBMCs) were isolated from total blood using Ficoll separation (GE Healthcare, ref 17-1440-02) and cryopreserved in liquid nitrogen until RNA isolation. Synovial tissue biopsies were collected by needle arthroscopy as previously described15 (link) from an inflamed knee as judged by the treating physician and cryopreserved in liquid nitrogen until RNA isolation.

Pollastro S., Klarenbeek P.L., Doorenspleet M.E., van Schaik B.D., Esveldt R.E., Thurlings R.M., Boumans M.J., Gerlag D.M., Tak P.P., Vos K., Baas F., van Kampen A.H, & de Vries N. (2019). Non-response to rituximab therapy in rheumatoid arthritis is associated with incomplete disruption of the B cell receptor repertoire. Annals of the Rheumatic Diseases, 78(10), 1339-1345.

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Isolation and Purification of Wound Macrophages

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MACs sorting of wound cell isolates was performed (Mirza et al., 2013 (link)). Briefly, wound cell isolates were incubated with fluorescein isothiocyanate (FITC)-labeled anti-mouse anti-CD3 (RRID:AB_312660), anti-NK1.1 (RRID:AB_448547), anti-CD19 (RRID:AB_2629813), and anti-Ly6G (Biolegend, RRID:AB_470400) monoclonal antibodies conjugated to FITC. Wound isolates were then washed and incubated with anti-FITC microbeads (Miltenyi Biotec, RRID:AB_244371, cat No. 130-049-601) and passed through a MACs column (Miltenyi Biotec). The resultant eluent was then incubated with anti-mouse anti-CD11b microbeads, (Miltenyi Bioter, cat No. 130-049-601). The remaining cell population was analyzed by flow cytometry and found to be 97% macrophages consistent with previous literature (Gallagher et al., 2015 (link); Mirza et al., 2013 (link)). For human monocyte isolation, peripheral blood was collected and subjected to RBC lysis and Ficoll separation (GE healthcare). Cell suspensions were then treated with anti-human CD14 microbeads. Magnetic separation yielded 95% purity by flow cytometry.

Kimball A.S., Davis F.M., denDekker A., Joshi A.D., Schaller M.A., Bermick J., Xing X., Burant C.F., Obi A.T., Nysz D., Robinson S., Allen R., Lukacs N.W., Henke P.K., Gudjonsson J.E., Moore B.B., Kunkel S.L, & Gallagher K.A. (2019). The Histone Methyltransferase Setdb2 Modulates Macrophage Phenotype and Uric Acid Production in Diabetic Wound Repair. Immunity, 51(2), 258-271.e5.

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Feline and Murine IFNγ ELISPOT Assay

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Mouse splenocytes or feline PBMCs isolated by ficoll separation (GE Healthcare) were maintained in Roswell Park Memorial Institute medium supplemented with fetal bovine sera, penicillin/streptomycin. Feline PBMCs were supplemented with 0.5 ng/mL feline IL2 (R&D Systems). Cells were plated at 2x10⁵/well in round bottom 96-well plates and cultured with 10 μg/mL feHER2-Fc (3T3 supernatant-equivalent as described above), huHER2-Fc, human IgG control (Jackson Immunolabs) or control 3T3-conditioned medium for 48 (mouse) or 72 (feline) hours. Total well contents were then transferred to mouse or feline (R&D Systems) IFNγ ELISPOT plates and incubated for an additional 48 hours prior to detection and enumeration as previously described (44 (link)) or per manufacturer protocol. Visualized cytokine spots were enumerated using the ImmunoSpot analyzer (CTL, Shaker Heights, OH) and expressed as the number of cytokine-producing cells per 10⁶ splenocytes or PBMCs.

Gibson H., Veenstra J., Jones R., Vaishampayan U., Sauerbrey M., Bepler G., Lum L., Reyes J., Weise A, & Wei W.Z. (2015). Induction of HER2 Immunity in Outbred Domestic Cats by DNA Electrovaccination. Cancer immunology research, 3(7), 777-786.

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Peripheral Blood Mononuclear Cells in Tuberculosis

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Sixty patients with TB and 60 healthy volunteers were recruited in this study at the Third People's Hospital of Kunming City during 2015‐2017. The diagnostic criteria for TB were TB symptoms by a chest X‐ray alone with positivity for M tb culture. The TB patients had no immune deficiency disease, hepatitis B, diabetes or other pulmonary‐related complications. Healthy controls were recruited from volunteers who had a routine physical examination at Third People's Hospital of Kunming City. The peripheral blood were collected into ETDA‐treated tubes from patients when admitted to the hospital for the first time. The PBMCs were isolated from the peripheral blood via density gradient centrifugation by Ficoll separation (GE Healthcare, Chicago, USA). After washing with PBS, the PBMCs were then subjected to further analysis. The study protocol was approved by the Institutional Ethics Committee Board of the Third People's Hospital of Kunming City, and the informed consent was obtained from each volunteer.

Li X., Huang S., Yu T., Liang G., Liu H., Pu D, & Peng N. (2019). MiR‐140 modulates the inflammatory responses of Mycobacterium tuberculosis‐infected macrophages by targeting TRAF6. Journal of Cellular and Molecular Medicine, 23(8), 5642-5653.

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