PBMCs were isolated with Ficoll-Hypaque density centrifugation (Sigma Aldrich, St Louis, Mo). TH17 cells were identified by means of intracellular staining of CD4+ T cells for the production of IL-17. Briefly, 1 × 106 cells from patients and an age matched healthy control subject were stimulated for 6 h with 10 ng/ml phorbol 12-myristate 13-acetate and 1 ug/ml ionomycin (Sigma-Aldrich, St Louis, Mo) in the presence of GolgiPlug (BD Biosciences, San Jose, CA). After cell-surface staining with PerCP-conjugated anti-CD4 (BD Biosciences, San Jose, CA), cells were fixed, permeabilized (Cytofix/Cytoperm, BD Biosciences, San Jose, CA), and stained with Alexa Fluor 647-conjugated anti-IL-17A (BD Biosciences, San Jose, CA). Immunoglobulin isotype control was used as a background control. CD4+ T cells were also evaluated for IFN-γ production (FITC-conjugated anti-IFN-γ; BD Biosciences, San Jose, CA). CD4+IL17+IFN-γ− cells were taken as TH17 cells (Supplementary Figure 1A ).
Ficoll hypaque density centrifugation
Manufactured by Merck Group
Sourced in United States
Ficoll-Hypaque density centrifugation is a laboratory technique used for the isolation and purification of cells and other biological materials. It involves the use of a density gradient medium, Ficoll-Hypaque, to separate different cellular components based on their density during centrifugation.
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Lab products found in correlation
Percp conjugated anti cd4, by BD (1 mentions)
Fitc conjugated anti ifn γ, by BD (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Jsh 23, by Abcam (1 mentions)
Ly364947, by Abcam (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
X vivo 15 medium, by Lonza (1 mentions)
U0126, by Abcam (1 mentions)
Texmacs medium, by Thermo Fisher Scientific (1 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
Human il 2, by Thermo Fisher Scientific (1 mentions)
Il 15, by Thermo Fisher Scientific (1 mentions)
Texmacs medium, by Miltenyi Biotec (1 mentions)
4 protocols using ficoll hypaque density centrifugation
1
Identification of TH17 Cells from PBMCs
2
Profiling Immune Cell Subsets
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PBMCs were isolated using Ficoll–Hypaque density centrifugation (Sigma, St Louis, MO, USA). Cells were divided into three groups for staining respectively including CD19, CD3/CD4/CD8, and CD14/CD16/CD56 and then incubated on ice for 40 min. After being washed with PBS containing 1% FBS for three times, cells were detected using the flow cytometer.
3
Macrophage Isolation and Stimulation
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Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque density centrifugation (Sigma, St Louis, MO, USA). Macrophages were isolated from the PBMCs using CD14+isolation MicroBeads (Miltenyi Biotec, Inc., Auburn, CA, USA). Isolated macrophages were cultured with X-vivo 15 medium (Lonza, Walkersville, MD, USA). For LPS (Sigma, St Louis, MO, USA) single stimulation, macrophages from HCV patients or healthy donors were treated with 100 ng ml−1 LPS. For HCV antigen and LPS double stimulations, macrophages were first incubated with HCV supernatant (from pJFH1-transfected Huh7.5 cells, with 108 plaque-forming units (p.f.u.) of HCVcc [12 (link)]) for 5 days and then treated with LPS stimulation. A20 adenovirus overexpression vectors (ad-A20) were purchased from Genechem Co. Ltd (Shanghai, PR China). Signalling inhibitors, including U0126, JSH-23 and LY364947 (Abcam, Cambridge, MA, USA), were added to the culture medium.
4
Expansion and Transduction of Activated T Cells
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Peripheral blood mononuclear cells (PBMCs) were prepared from buffy coats of healthy donors after receiving approval (507–1997) from the Institutional Review Board of the University of Florida (IRB-01). PBMCs were isolated by Ficoll-Hypaque density centrifugation (Sigma Aldrich, St Louis, MO, USA) . PBMCs were activated with phytohemagglutinin (PHA; 5 µg/ml) for 2–3 days, after which they were maintained in TexMACS™ medium (Miltenyi Biotec Inc, San Diego, CA, USA). Activated T lymphocytes were transduced with CD19- or GD2-specific lentiviral particles, and were then maintained in TexMACS™ medium supplemented with human IL-2 (40 U/ml), IL-7 (20 U/ml), and IL-15 (10 U/ml) (PeproTech, Cranbury, CT, USA). Cells were expanded over 7–10 days in the presence of cytokines. After expansion, 1 × 105 cells were extracted for genomic DNA and subjected to real-time PCR analyses to determine the viral gene copy number.
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