Ficoll hypaque plus gradient

Manufactured by GE Healthcare

Sourced in France, United States

Ficoll-Hypaque Plus gradient is a separation medium used for the isolation of mononuclear cells from whole blood or other cellular samples. It is a density gradient centrifugation solution designed to facilitate the separation of different cell types based on their density.

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Lab products found in correlation

Efluor 450 conjugated anti cd3, by Thermo Fisher Scientific (1 mentions) Cd66abce microbead kit, by Miltenyi Biotec (1 mentions) Apc conjugated anti cd14, by BD (1 mentions) Aim 5, by Thermo Fisher Scientific (1 mentions) Srt1720, by Selleck Chemicals (1 mentions) Facscalibur, by BD (1 mentions) Cellquest software, by BD (1 mentions) Dithiothreitol (dtt), by AppliChem (1 mentions) Collagenase and dnase, by Merck Group (1 mentions)

Spelling variants of this product

Ficoll-Hypaque (553 citations) Ficoll (495 citations) Ficoll-Hypaque gradient (102 citations) Ficoll gradient (93 citations) Ficoll-Hypaque Plus (48 citations) Ficoll-Plus gradient (5 citations)

3 protocols using ficoll hypaque plus gradient

Purification of Immune Cell Populations

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Fresh heparinized whole blood from healthy donors was utilized for neutrophil, CD14, and CD2 purifications. Whole blood was lysed (155 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA in distilled water) for 10 min at RT and washed twice for 10 min at 300 × g. CD66b⁺ neutrophils were isolated by positive selection using the CD66abce Microbead kit (Miltenyi Biotec) according to manufacturer’s protocol. Purity was assessed by flow cytometry using BV421-conjugated anti-CD66b and APC-conjugated anti-CD14 (BD Biosciences). The purity of the enriched population was >98%. CD14⁺ and CD2⁺ cells were positively isolated from the PBMC fraction obtained by Ficoll-Hypaque Plus gradient separation (GE Healthcare, France), using CD14 and CD2 Microbeads (Miltenyi Biotec), according to manufacturer’s protocol. Briefly, CD14⁺-labeled cells were first enriched on positive selection columns and the depleted fraction containing total lymphocytes was washed and stained with CD2 Microbeads and isolated on positive selection columns. Purity of CD14 and CD2 enriched fractions was assessed by flow cytometry using APC-conjugated anti-CD14 (BD Biosciences) and efluor450-conjugated anti-CD3 (Ebioscience, France) and was >98%. Viability of the samples following each purification procedure was assessed by Trypan blue staining and was >97% for all experiments.

Bisiaux A., Boussier J., Duffy D., Quintana-Murci L., Fontes M, & Albert M.L. (2017). Deconvolution of the Response to Bacillus Calmette–Guérin Reveals NF-κB-Induced Cytokines As Autocrine Mediators of Innate Immunity. Frontiers in Immunology, 8, 796.

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PMNC Response to MG-BSA in Metabolic Syndrome

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PMNCs from individuals with the metabolic syndrome were separated from fasting, EDTA anticoagulated blood by Ficoll-Hypaque Plus gradient (GE Healthcare Bioscience, Pittsburgh, PA, USA) and incubated in serum-free culture media AIM-V (Invitrogen, Carlsbad, CA, USA) at 37°C with 5% CO₂ for 1 h, followed by the addition of MG-BSA (60 μg/ml) [12 (link), 14 (link), 16 (link)]. MG-BSA contained 22 MG-modified arginine residues per mole based on HPLC. MG-BSA was passed through an endotoxin-binding affinity column (Pierce, Rockford, IL, USA) to remove endotoxins [12 (link), 14 (link)] (ESM Methods). Cells were incubated with or without the SIRT1 inhibitor Sirtinol (10 μmol/l; Calbiochem, La Jolla, CA, USA) or a SIRT1 activator, SRT1720 (1 μmol/l; Selleckchem, Houston, TX, USA). After 72 h, the cells were harvested for western blotting, the culture medium was centrifuged at 1000 g for 10 min and the supernatant fraction was collected for testing human TNFα levels with an ELISA kit (Invitrogen) [28 (link)]. In a separate study, PMNCs from normal volunteers without the metabolic syndrome, described previously [20 (link)], were used as controls for PMNCs from individuals with the metabolic syndrome. All baseline cellular data were collected from PMNCs obtained at study commencement.

Vlassara H., Cai W., Tripp E., Pyzik R., Yee K., Goldberg L., Tansman L., Chen X., Mani V., Fayad Z.A., Nadkarni G., Striker G.E., He J.C, & Uribarri J. (2016). Oral AGE restriction ameliorates insulin resistance in obese individuals with the metabolic syndrome: a randomised controlled trial. Diabetologia, 59(10), 2181-2192.

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Isolation and Phenotyping of Gut Immune Cells

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Single-cell suspensions were obtained by enzymatic digestion of mucosal specimens and subjected to flow cytometry to determine the proportion of epithelial cells (Cytokeratin, CK+) acting as APCs (expressing CD80, HLA-ABC) and the proportion of activated CD8+ T cells (positive for CD28 and CD38). Esophageal mucosa was stripped from the muscularis mucosa, cut into strips, and freed of mucus by a 30-min wash in Hank's balanced salt solution (HBSS) containing 10 mM dithiothreitol (DTT; AppliChem). Intestinal epithelial cells (IEC) were isolated with a 30-min incubation of the intestinal mucosa in HBSS containing 1 mM ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich). For lamina propria mononuclear cells (LPMCs), stripped mucosa, freed of mucus and IEC, was digested with 1 mg/mL collagenase and DNase (Sigma-Aldrich) for 30 min at 37°C. The resulting crude cell suspensions were purified using a Ficoll-Hypaque Plus gradient (GE Healthcare), and the preparations were preferentially enriched for LPMC, washed, and collected.
Flow cytometric analysis was performed using a FACSCalibur based on CellQuest software (Becton Dickinson). The antibodies used are summarized in Supplementary Table 1.

Scarpa M., Kotsafti A., Fassan M., Scarpa M., Cavallin F., Nardi T., Pinto E., Alfieri R., Cagol M., Agostini M., Rugge M., Castagliuolo I, & Castoro C. (2017). Immunonutrition before esophagectomy: Impact on immune surveillance mechanisms. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, 39(10).

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