Ficoll hypaque density gradient centrifugation

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Ficoll-Hypaque density gradient centrifugation is a laboratory technique used for the separation and purification of cells and cellular components based on their density. It involves the use of a density gradient medium, Ficoll-Hypaque, to isolate specific cell types or subcellular fractions by centrifugation.

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Lab products found in correlation

Percoll density gradient centrifugation, by GE Healthcare (2 mentions) Rhgm csf, by Thermo Fisher Scientific (2 mentions) Rhil 4, by Thermo Fisher Scientific (2 mentions) Lipopolysaccharides (lps), by Merck Group (2 mentions) L glutamine penicillin streptomycin solution, by Merck Group (1 mentions) Human glioma cell lines, by American Type Culture Collection (1 mentions) Ccl21, by Thermo Fisher Scientific (1 mentions) Cxcl11, by Thermo Fisher Scientific (1 mentions)

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Ficoll-Hypaque (32 citations) Ficoll (27 citations) Density gradient centrifugation (7 citations) Ficoll density gradient centrifugation (4 citations) Ficoll-Hypaque gradient centrifugation (2 citations)

7 protocols using ficoll hypaque density gradient centrifugation

Isolation and Transfection of PBMCs

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30 mL of peripheral blood of the patients and healthy subjects was collected and then added with heparin anticoagulation (20 U/mL) for next experiments. Ficoll-Hypaque density gradient centrifugation (Solarbio, Beijing, China) was performed for PBMCs isolation. RPMI 1640 or DMEM was respectively used for cell culture of PBMCs and 293T, and 10% fatal bovine serum was used for maintaining cell growth. Besides, 100 g/mL penicillin G and streptomycin were also added into the medium to maintain cell growth. All cells were cultured in an incubator with 37°C and 5% CO₂.
For cell transfection, when cellular confluence was at 70%, the 500 μl serum-free medium containing 10 μl Lipofectamine 2000 (Beijing Noble Ryder Technology Co., Ltd., Beijing, China) and 4 μg of DNA or100 pmol RNA were added into each well. Finally, the cells were further cultured for 24 hours.

Luo S., Wu R., Li Q, & Zhang G. (2021). MiR-301a-3p Advances IRAK1-Mediated Differentiation of Th17 Cells to Promote the Progression of Systemic Lupus Erythematosus via Targeting PELI1. Journal of Healthcare Engineering, 2021, 2982924.

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Isolation of Peripheral Blood Mononuclear Cells

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Whole blood was centrifuged for 20 min at 2000 rpm. The pelleted cells were resuspended in PBS at a ratio of 1 : 1 and were separated by Ficoll-Hypaque density gradient centrifugation (Solarbio, Beijing, China) for 30 min at 2000 rpm. The cells in the white layer were collected, resuspended in 50 ml of PBS, and centrifuged for 15 min at 1500 rpm. The pelleted cells were resuspended in 50 ml of PBS and centrifuged for 15 min at 1300 rpm. The PBMC pellet was obtained after discarding the supernatant. The mononuclear cells were resuspended in 50 ml of PBS, counted using a hemocytometer, and examined with trypan blue dye solution.

Liu C., Zheng Y., Tang J., Wang D., Ma Z., Li S, & Chang X. (2019). Stimulation of DC-CIK with PADI4 Protein Can Significantly Elevate the Therapeutic Efficiency in Esophageal Cancer. Journal of Immunology Research, 2019, 6587570.

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PBMC Isolation from Healthy Volunteers

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Venous blood was taken from the healthy volunteers, and the samples were EDTA‐anticoagulated. The anticoagulated blood was treated by Ficoll–Hypaque density gradient centrifugation (Solarbio, P9011), and the PBMCs were separated. PBMCs were washed and resuspended in RPMI 1640 medium, supplemented with 10% FBS, at a concentration of 1.5 × 10⁶ cells/ml for investigation. Ethical approval and informed consent for each donor were granted by the ethics committee of Chinese PLA General Hospital (S2021‐046‐01).

Chen H., Yang T., Xu Y., Liang B., Liu X, & Cai Y. (2024). Anti‐inflammatory and immunoregulatory effects of colistin sulphate on human PBMCs. Journal of Cellular and Molecular Medicine, 28(8), e18322.

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Generation of Monocyte-Derived Dendritic Cells

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Human peripheral blood mononuclear cell (PBMC) were isolated from the peripheral blood of healthy donors at the Blood Centre of Anhui Province using Ficoll-Hypaque density gradient centrifugation (Solarbio, Beijing, China). Subsequent Percoll density gradient centrifugation (GE Healthcare, Chicago, IL, USA) was performed for monocyte separation as previously reported54 (link). Human monocyte-derived DCs were generated after culturing the monocytes with rhGM-CSF (50 ng/mL, PeproTech, Rocky Hill, NJ, USA) and rhIL-4 (50 ng/mL, PeproTech) in RPMI 1640 medium (RPMI 1640 with 2 mM glutamine, 25 mM HEPES, 100 U/ml penicillin, and 100 mg/ml streptomycin) with the addition of sera from cancer patients or healthy controls, followed by incubation at 37 °C in 5% CO₂ for 5 days. To obtain mature DCs, the cells were cultured in the same cytokine mixtures in the presence of LPS (50 ng/mL, Sigma-Aldrich, St. Louis, MO, USA) for an additional 2 days.

Li R., Fang F., Jiang M., Wang C., Ma J., Kang W., Zhang Q., Miao Y., Wang D., Guo Y., Zhang L., Guo Y., Zhao H., Yang D., Tian Z, & Xiao W. (2017). STAT3 and NF-κB are Simultaneously Suppressed in Dendritic Cells in Lung Cancer. Scientific Reports, 7, 45395.

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CD8+ T Cell Culture with CHI3L2

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The human glioma cell lines and a human microglia cell line were purchased from the American Type Culture Collection resource center. The human glioma cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and the human microglia cells were cultured in Minimum Essential Medium (MEM) with 10% FBS at 37 °C in a humidified incubator containing 5% CO2. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation (Solarbio, Beijing, China). CD8+ T cell were separated by positive selection from PBMCs with CD8 magnetic beads and cultured in RPMI-1640 medium supplemented with 10% human serum, 5% L-glutamine-penicillin-streptomycin solution (Sigma-Aldrich, USA), CD3/CD28 antibody (Biolegend, USA) (25ul/ml) and IL-2 (100IU/ml) in 24-well plates. After culturing for 24 hours, add the corresponding concentration of human CHI3L2 protein (Sino Biological, Beijing, China) to T cells and culture for 72 hours at 37 °C in a humidified incubator containing 5% CO2.

Liu L., Yang Y., Duan H., He J., Sun L., Hu W, & Zeng J. (2021). CHI3L2 Is a Novel Prognostic Biomarker and Correlated With Immune Infiltrates in Gliomas. Frontiers in Oncology, 11, 611038.

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Generation of Human Monocyte-Derived Dendritic Cells

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Human PBMCs were isolated from the peripheral blood of healthy donors by Ficoll-Hypaque density gradient centrifugation (Solarbio). Subsequent Percoll density gradient centrifugation (GE Healthcare) was performed for monocyte separation as previously reported [29] (link). Human monocyte-derived DCs were generated by culturing the cells with rhGM-CSF (50 ng/mL, PeproTech) and rhIL-4 (50 ng/mL, PeproTech) in RPMI 1640 medium (RPMI 1640 with 2 mM glutamine, 25 mM HEPES, 100 U/ml penicillin, and 100 mg/ml streptomycin) that contained 10% fetal bovine sera (HyClone) at 37°C in a CO₂ (5%) incubator for 5 days. To obtain mature DCs, the cells were cultured in the same cytokine mixtures in the presence of LPS (50 ng/mL, Sigma-Aldrich) for an additional 2 days.

Guo Y., Li R., Song X., Zhong Y., Wang C., Jia H., Wu L., Wang D., Fang F., Ma J., Kang W., Sun J., Tian Z, & Xiao W. (2014). The Expression and Characterization of Functionally Active Soluble CD83 by Pichia pastoris Using High-Density Fermentation. PLoS ONE, 9(2), e89264.

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Expansion and Activation of Cytotoxic T Cells

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Blood samples from healthy donors or patients with CRC were processed using Ficoll-Hypaque density gradient centrifugation (Beijing Solarbio Science and Technology Co., Ltd.) to obtain PBMCs. After washing in RPMI-1640 medium, 2×10⁶ cells were resuspended in RPMI-1640 medium containing 10% FBS, 2 mM glutamine, 100 IU/ml penicillin and 100 IU/ml streptomycin in a cell culture flask. After incubation for 24 h with 1,500 IU/ml INF-γ (Shanghai ChemoWanbang Biopharma Co., Ltd.; cat. no. S10980084), 5,000 IU/ml IL-2 (Beijing T&L Biotechnology Co., Ltd; cat. no. TL-104) and 100 ng/ml anti-CD3 antibodies (1:10,000; Wuhan Institute of Biological Products Co., Ltd.; cat. no. S19990012) were added and maintained at 37°C in humidified atmosphere of 5% CO₂ for 2 days. Then fresh culture medium containing 5,000 IU/ml recombinant human (rh)IL-2 was added every 2 to 3 days. In some assays, the recombinant chemokine ligand CCL21 (100 ng/ml) or CXCL11 (10 ng/ml) (PeproTech, Inc.) was added during the culture process. The cells were maintained at 37°C in a humidified atmosphere of 5% CO₂. After 13 days, cells were harvested for flow cytometry or Transwell analysis.

Zou Y., Liang J., Li D., Fang J., Wang L., Wang J., Zhang J., Guo Q., Yan X, & Tang H. (2020). Application of the chemokine-chemokine receptor axis increases the tumor-targeted migration ability of cytokine-induced killer cells in patients with colorectal cancer. Oncology Letters, 20(1), 123-134.

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