Ficoll density gradient

Manufactured by Merck Group

Sourced in United States, Germany

Ficoll density gradient is a laboratory technique used for the separation and purification of cells, cellular organelles, and other biological macromolecules. It utilizes a gradient of Ficoll, a synthetic, highly branched, and hydrophilic polymer, to facilitate the separation of different components based on their density.

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Ficoll (251 citations) Ficoll gradient (46 citations) Ficoll-Hypaque density gradient (36 citations) Ficoll density gradient centrifugation (22 citations) Ficoll-Hypaque density gradient centrifugation (13 citations) Ficoll Histopaque density gradient (9 citations) Ficoll density gradient separation (6 citations) Ficoll-Paque Plus density gradient (6 citations) Ficoll-Paque density gradient (6 citations) Ficoll-Paque density gradient centrifugation (5 citations)

20 protocols using ficoll density gradient

Isolation and Purification of VZV DNA from PBMCs

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Peripheral blood mononuclear cells (PBMCs) were separated from fresh whole blood by Ficoll density gradient centrifugation (Sigma, Germany), re-suspended in RPMI (Sigma, Germany), and stored in liquid nitrogen for future use. VZV DNA was extracted from the PBMC samples using an extraction kit (Qiagen, Germany), according to the manufacturer’s instructions. The purity of the extracted DNA was confirmed based on the absorbance of the extracted DNA at 260 nm and 280 nm wavelengths by biophotometer (Eppendorf, Germany). The samples were stored in a -80°C freezer for future tests.

Najafi S., Ghane M., Yousefzadeh-Chabok S, & Amiri M. (2016). The High Prevalence of the Varicella Zoster Virus in Patients With Relapsing-Remitting Multiple Sclerosis: A Case-Control Study in the North of Iran. Jundishapur Journal of Microbiology, 9(3), e34158.

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Mononuclear Cell Isolation and Methylation Sequencing

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For the enrichment of mononuclear cells, bone marrow samples were separated using a Ficoll density gradient (Sigma-Aldrich, St. Louis, MO, USA), and DNA from the lymphoblasts was purified using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). MethylC-capture sequencing was performed as described previously [13 (link)]. Briefly, an amplified bisulfate-converted DNA fragment library was constructed using a SeqCap Epi enrichment system (Roche NimbleGen, Madison, WI, USA), and a HiSeq2500 system (Illumina, San Diego, CA, USA) was used for DNA sequencing.

He S., Li Y., Shi X., Wang L., Cai D., Zhou J, & Yu L. (2023). DNA methylation landscape reveals LIN7A as a decitabine-responsive marker in patients with t(8;21) acute myeloid leukemia. Clinical Epigenetics, 15, 37.

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Isolation and Culture of GFP-Expressing Rat Mononuclear Cells

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The peripheral blood was harvested from green fluorescence protein (GFP) transgenic Sprague-Dawley rats (“green rat CZ-004” SD TgN(act-EGFP) OsbCZ-004), aged 4 weeks, and diluted immediately with heparinized saline in a 1:1 proportion. The diluted blood was gently loaded onto Ficoll density gradient (Sigma) in 10 mL tubes and centrifuged for 25 min at 1600 g. Mononuclear cell fraction was collected and rinsed twice with Hank’s Balanced Salt Solution (HBSS), and then cultured at 10⁶ cells/cm² in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS). The medium was replaced every three days and non-adherent cells were discarded. After the primary culture reached approximately 80% confluence, cells were passaged regularly and the 5^th passaged (P5) cells were used for further experiments in this project.

Wu G., Pan M., Wang X., Wen J., Cao S., Li Z., Li Y., Qian C., Liu Z., Wu W., Zhu L, & Guo J. (2015). Osteogenesis of peripheral blood mesenchymal stem cells in self assembling peptide nanofiber for healing critical size calvarial bony defect. Scientific Reports, 5, 16681.

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Differentiation of M2-like Macrophages from Human PBMCs

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Human PBMCs were collected from the venous blood of healthy volunteers, and in leukocyte reduction chambers, separated via ficoll density gradient (Sigma, St. Louis, MO, USA). CD14⁺ monocytes were positively selected to >95% purity by magnetic activated cell sorting using anti-CD14 microbeads (Miltenyi, USA). For the induction of macrophage differentiation, the sorted CD14⁺ monocytes were cultured in RPMI 1640 supplemented with 10% FBS and 20 ng/mL human M-CSF (PeproTech, USA). Fresh medium with M-CSF was added every 3 days. On day 7, M2-like polarization was achieved by treatment with human 20 ng/mL IL-4 and 20 ng/mL IL-13 (PeproTech, USA) for 24 h. For M0, only PRMI 1640 + 10% FBS was added.

Zhang W., Xu L., Zhang X., Xu J, & Jin J.O. (2023). Escherichia coli adhesion portion FimH polarizes M2 macrophages to M1 macrophages in tumor microenvironment via toll-like receptor 4. Frontiers in Immunology, 14, 1213467.

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Isolation of Leukemic Cells from ALL Patients

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After written informed consent was obtained, leukemic cells were gathered from ALL patients with CNS metastasis. By separating mononuclear cells on a Ficoll density gradient (Sigma-Aldrich, St. Louis, MO, USA), cell suspensions containing >80% leukemic cells were produced. The present study was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology, and performed in strict compliance with the Declaration of Helsinki.

Li Z., Guo Z., Xiao H., Chen X., Liu W, & Zhou H. (2024). Simulating neuronal development: exploring potential mechanisms for central nervous system metastasis in acute lymphoblastic leukemia. Frontiers in Oncology, 13, 1331802.

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Isolation and Cryopreservation of Human BMSCs

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Primary human BMSCs were derived from identified human whole bone marrow aspirates. Usage of these samples has been approved by the Institutional Review Board of The Methodist Hospital Research Institute (TMHRI) and Wake Forest University Health Science (WFUHS). BMSCs isolation was conducted as previously described [26 (link)]. Bone marrow mononuclear cells were obtained by Ficoll density gradient (1.077 g/mL; Sigma, St. Louis, MO) and then plated into 35 cm² tissue culture flasks at a concentration of 10⁶ cells/mL in Mesencult basal medium supplemented with MSC stimulatory supplements (Invitrogen, Vancouver, BC). After 24h incubation at 37°C in a 5% CO₂ humidified atmosphere, non-adherent cells were removed, and the adherent fraction was cultured with fresh medium. When reaching 60% confluence, cells were trypsinized (0.25% trypsin/EDTA, Gibco, Vancouver, BC) and replated into a 100 cm² tissue culture dish. The cells at fourth passages were cryopreserved in a liquid nitrogen tank and stored for subsequent experiments. The human marrow stromal cell lines HS5, derived from the marrow of a healthy volunteer were obtained from ATCC.

Wu D., Guo X., Su J., Chen R., Berenzon D., Guthold M., Bonin K., Zhao W, & Zhou X. (2014). CD138-negative myeloma cells regulate mechanical properties of bone marrow stromal cells through SDF-1/CXCR4/AKT signaling pathway. Biochimica et biophysica acta, 1853(2), 338-347.

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Isolation of T Cell Subsets from Gastric Cancer Patients

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Fresh peripheral blood was collected in sodium-heparin vacutainer tubes. Periphery mononuclear cells (PBMCs) were isolated by Ficoll density gradient (Sigma Aldrich) centrifugation. CD8⁺ T, TCRγδ⁺ T, CD4⁺CD25⁻ T, and CD4⁺CD25⁺ Treg cells were isolated and purified from fresh PBMCs of patients with gastric cancer by magnetic cell separation. In brief, CD8⁺ T (>95% purity) and TCRγδ⁺ T cells (>95% purity, named as peripheral-derived γδ T cells) were firstly separated by positive selection using human blood TCRγδ⁺ T and CD8⁺ T Cell Isolation Kits (Miltenyi Biotec) according to the manufacturer's instructions. From the fraction of remaining cells further isolation of CD4⁺CD25⁻ T cells (>90% purity) and CD4⁺CD25⁺ T cells (>95% purity) was accomplished by negative selection and positive selection using a human CD4⁺CD25⁺ Regulatory T Cell Isolation Kit (Miltenyi Biotec).

Mao C., Mou X., Zhou Y., Yuan G., Xu C., Liu H., Zheng T., Tong J., Wang S, & Chen D. (2014). Tumor-Activated TCRγ δ + T Cells from Gastric Cancer Patients Induce the Antitumor Immune Response of TCRα β + T Cells via Their Antigen-Presenting Cell-Like Effects. Journal of Immunology Research, 2014, 593562.

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HTLV-1 Proviral Load Quantification

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Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA-treated blood samples by Ficoll density gradient (Sigma, Germany). DNA was extracted using Ge Net kit (Korea). To assess the HTLV-1 proviral load, a real-time TaqMan PCR absolute method was carried out using HTLV-1 RG kit (Novin Gene, Iran) (28 (link)). Briefly, the HTLV-1 copy number was referred to the actual amount of cellular DNA by quantifying albumin gene as the reference gene. HTLV-1and albumin DNA concentrations were calculated using two 5-point standard curves. The normalized value of the HTLV-1 proviral load was calculated as the ratio of (HTLV-1 DNA copies number /albumin DNA copies number/2) ×10⁴ and expressed as the number of HTLV-1 proviruses per 10⁴ PBMCs (7 (link)).

Yari A., Rezaee S.A., Valizadeh N., Rajaee T., Jazayeri S.M., Soltani M, & Norouzi M. (2014). Evaluation of HTLV-1 activity in HAM/TSP patients using proviral load and Tax mRNA expression after In Vitro lymphocyte activation. Iranian Journal of Basic Medical Sciences, 17(7), 531-536.

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Isolation and Differentiation of Human Monocytes

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Human blood samples were collected into vacutainer EDTA tubes (Greiner Bio-One, Mosonmagyarovar, Hungary). PBMCs (peripheral blood mononuclear cells) were isolated via a Ficoll density gradient (Sigma, St. Louis, MO, USA). CD14+ cells were isolated from PBMCs using a positive magnetic selection method (EasySep, StemCell Technologies, Vancouver, Canada) (29 (link)). After isolation, a third of the monocytes were immediately frozen in liquid nitrogen. Two-thirds of monocytes were cultured (1x10⁵ monocytes per well) in MEM α (Minimum Essential Medium α) (Sigma, St. Louis, MO, USA) with 10% FBS (Gibco Laboratories, Gaithersburg, MD, USA), 1% L-glutamine (Sigma, St. Louis, MO, USA), and 1% Penicillin–Streptomycin (Sigma, St. Louis, MO, USA) at 37°C and 5% CO₂ in a humidified atmosphere. Monocytes were incubated with 50 ng/mL rh M-CSF (macrophage colony-stimulating factor) for 24 h (PeproTech, London, UK). Thereafter the samples were stimulated with 50 ng/mL of both rh M-CSF and rh RANKL (receptor activator of nuclear factor κB ligand) (PeproTech, London, UK). From then the media was replaced every 3-4 days.

Kovács O.T., Tóth E., Ozohanics O., Soltész-Katona E., Marton N., Buzás E.I., Hunyady L., Drahos L., Turu G, & Nagy G. (2022). Proteomic Changes of Osteoclast Differentiation in Rheumatoid and Psoriatic Arthritis Reveal Functional Differences. Frontiers in Immunology, 13, 892970.

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HTLV-1 Proviral Load Quantification

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Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA-treated blood samples by Ficoll density gradient (Sigma, Germany). A quantitative real-time PCR assay (TaqMan method) was carried out to measure Tax expression and proviral load of HTLV-1 in PBMCs using specific primers and a fluorogenic probe by a Rotor Gene Q real-time PCR machine. RNA was extracted from fresh PBMCs using viral RNA extraction Mini Kit (Qiagen, Germany) and complementary DNA (cDNA) was synthesized using Revert Aid™ First Strand cDNA Synthesis Kit (Fermentas, Germany). Table 1 shows the nucleotide sequence of primers and probes. The procedure for conventional PCR was as followes: 1 µl cDNA (288 ng/µl), 2.5 µl reaction buffer 28x, 8.5 µl dNTP (10 mM, GeNet Bio), 2 µl MgCl₂ (25 mM, Fermentas), 0.5 µl each of primers (28 pM), 0.2 µl prime Taq DNA polymerase (5 U/µl, Fermentas), 12.8 µl distilled water. β₂m was used as human housekeeping or reference gene in Tax expression measurement. The expression of this gene remains stable within PBMCs from samples (27 (link)).

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