PBMC were isolated from heparinized venous blood by Ficoll-Hypaque gradient centrifugation (GE Healthcare, Uppsala, Sweden) and diluted in RPMI medium supplemented with 10% AB human serum (Sigma, St Louis, MO,U.S.A.). PBMC cultures (2.0 × 105cells/well) were incubated in 96-well microplates (Costar, Cambridge, MA, U.S.A.) in medium in the presence of ligands for TLR2 (pam3csk4, 0.5 gmL−1); TLR3 [poly(I:C)-RIG, 20 (link) ng mL −1]; TLR4 (LPS, 1.25 μg mL −1); TLR5 (flagellin, 0.5 ng mL −1); TLR7 (imiquimod, 1.25 gmL−1); TLR7/TLR8 (CL097, 2.5 μg mL−1) and TLR9 (CpGA 2206,4 mol L−1) for 48 h at 37°C and 5% CO2. All ligands were obtained from InvivoGen (San Diego, CA, U.S.A.). Cell-free supernatants were harvested and stored at −80°C until cytokine measurements were obtained with a cytometric bead array.
Ficoll hypaque gradient centrifugation
Manufactured by GE Healthcare
Sourced in Sweden
Ficoll-Hypaque gradient centrifugation is a laboratory technique used for the separation and isolation of different types of cells, such as lymphocytes, from a mixed cell population. It utilizes a density gradient medium to separate cells based on their buoyant density during centrifugation.
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Lab products found in correlation
Human ab serum, by Merck Group (2 mentions)
96 well microplate, by Corning (1 mentions)
Dimethyl sulphoxide, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Edta buffered collection tubes, by Sarstedt (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
L2630, by Merck Group (1 mentions)
Anti cd3, by BD (1 mentions)
Anti cd28, by BD (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Rnalater solution, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Flt3l, by Thermo Fisher Scientific (1 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions)
Pyrilamine, by Merck Group (1 mentions)
Cl097, by InvivoGen (1 mentions)
Histamine, by Merck Group (1 mentions)
7 protocols using ficoll hypaque gradient centrifugation
1
Immune Response Profiling of PBMC Cells
2
Blood Collection and PBMC Isolation
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EDTA-buffered collection tubes (Sarstedt, Nümbrecht, Germany) were used for the collection of whole blood samples by venous puncture between 7 a.m. and 2 p.m. Data on the precise time of blood collection was available from a subcohort of N = 32 study participants (N = 14 MDD patients and N = 18 control subjects). In this subcohort, there was no significant group difference in the time of blood collection between the depressed and the control group (Table 1 ). Subsequent to blood drawings, peripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll-Hypaque gradient centrifugation according to the manufacturer’s protocol (GE Healthcare, Chalfon St Giles, UK) and isolated cells were stored frozen at −80 °C in cryoprotective freezing medium (dimethyl sulphoxide: Sigma-Aldrich, St. Louis, MO, USA; fetal calf serum: Sigma-Aldrich; dilution 1:10).
3
Immune Cell Proliferation Assay
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PBMCs were collected and purified from the peripheral blood of 6 healthy donors (3 males, 3 females) using Ficoll-Hypaque gradient centrifugation (GE Healthcare Life Sciences, Marlborough, MA, http://www.gelifesciences.com ). CD4+ T cells and CD19+ B cells were purified with CD4 and CD19 immunomagnetic bead antibodies, respectively, incubated with 5 mM carboxyfluorescein succinimidyl ester (CFSE, Thermo Fisher Scientific, Waltham, MA, USA, http://www.thermofisher.com ) for 15 min, washed with PBS containing 10% FBS, and then added to the coculture system with 2 mL medium at a ratio of 1:5 or 1:1 (MSCs [0.5 × 105 cells]:CD4+ T [2.5 × 105 cells] or CD19+ B cells [0.5 × 105 cells]). The cells were cocultured in RPMI-1640 medium containing purified anti-CD3 (0.2 mg/mL, BD Biosciences) and anti-CD28 (1 mg/mL, BD Biosciences) antibodies to stimulate the T cells and LPS (2.5 μg/mL, Sigma-Aldrich, L2630) to stimulate the B cells to proliferate for 5 days. CD4+ T cell and CD19+ B cell proliferation was measured by fluorescence-activated cell sorting.
4
Equine Mesenchymal Stem Cell Isolation
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Bone marrow aspirate was obtained from equine ilium, and mesenchymal stem cells (MSCs) were separated from the aspirate by density-gradient centrifugation (Ficoll-Hypaque gradient centrifugation) (GE Healthcare, Pittsburgh, PA, USA) and further cultured [22 (link)].
DOI;dx.doi.org/10.17504/protocols.io.kcicsue
DOI;
5
Isolation and culture of PBMCs
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PBMCs were isolated from heparinized venous blood by Ficoll–Hypaque gradient centrifugation (GE Healthcare, Uppsala, Sweden) and frozen in RNAlater ™ solution (Invitrogen, Waltham, MA, USA) for further analysis.
All cells were cultured in an RPMI-1640 medium (Sigma, St. Louis, MO, USA) with 1% penicillin/streptomycin (Sigma) at 37 °C with 5% CO2. The cell lines used in this study have been described elsewhere [41 (link),42 (link),43 (link)]. Hut78 was supplemented with 10% fetal bovine serum (FBS) (Sigma); meanwhile, SeAx and SeZ4 were supplemented with 10% AB human serum (Sigma) and 5 ng/mL of human recombinant IL-2 and IL-4 (Thermo Fisher Scientific, Waltham, MA, USA). The cell lines were a generous gift from Professor N. Ødum (Copenhagen, Denmark).
All cells were cultured in an RPMI-1640 medium (Sigma, St. Louis, MO, USA) with 1% penicillin/streptomycin (Sigma) at 37 °C with 5% CO2. The cell lines used in this study have been described elsewhere [41 (link),42 (link),43 (link)]. Hut78 was supplemented with 10% fetal bovine serum (FBS) (Sigma); meanwhile, SeAx and SeZ4 were supplemented with 10% AB human serum (Sigma) and 5 ng/mL of human recombinant IL-2 and IL-4 (Thermo Fisher Scientific, Waltham, MA, USA). The cell lines were a generous gift from Professor N. Ødum (Copenhagen, Denmark).
6
Isolation and Culture of Murine Hematopoietic Progenitor Cells
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Animal experimental procedures were performed with the approval of Lab Animal Ethics & Welfare committee of the Northwestern Polytechnical University. Wide-type female C57BL6 mice between 5 and 8 weeks of age were obtained from Laboratory Animal Center of the Xi'an Jiaotong University. Primary BM cell suspension was isolated by crushing and filtering the mice femurs and tibias marrow tissue through a 40-μm cell strainer. BM mononuclear cells were separated by Ficoll-Hypaque gradient centrifugation (density 1.084 g/ml, GE healthcare). Lin-c-kit+ HPCs were collected using magnetic activated cell sorting technology (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. The purity of the sorted cells was assessed by flow cytometry. Freshly isolated cells were either immediately analyzed or kept in IMDM medium supplemented with 10%FBS (Gibco), 50 ng/ml rm SCF, 20 ng/mL rm TPO and 20 ng/mL rm Flt-3L (all from PeproTech, Rocky Hill, NJ) for hydrogel culture.
7
Histamine and Toll-like Receptor Activation
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Mononuclear cells (MNCs) were isolated from cord blood and from venous blood of healthy adult individuals via Ficoll-Hypaque gradient centrifugation (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The cells were cultured in the presence of antagonists of H1R (pyrilamine, 100 μM), H2R (cimetidine, 1000 μM) and H4R (JNJ7777120, 1 μM) for 1 h and then with histamine (10 μM) (Sigma-Aldrich, St. Louis, MO, USA) and either a TLR-4 agonist (ultrapure lipopolysaccharide (LPS) from Salmonella minnesota, 1 μg/mL; endotoxin level ≤1 × 105 EU/mg) or a dual TLR7/TLR8 agonist (CL097, 2.5 μg/mL; Invivogen, San Diego, CA, USA) for 24 h. In some experiments, human IFN-γ recombinant (25 ng/mL, Peprotech, Rock Hill, NJ, USA) was added to LPS-stimulated cultures.
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