The PBMCs from the blood of the Duroc × Landrace × Large White (DLW) crossbred piglets (∼15 kg, ∼8 wk old) were isolated by Ficoll-Hypaque density gradient centrifugation at 25°C.12 (link) The PBMCs were cultured in RPMI 1640 medium (Gibco, Australia) supplemented with 10% heat-inactivated FBS (Gibco, Australia), 2 mmol/l l -glutamine, 100 U/ml penicillin and streptomycin (Gibco) at 37°C under 5% CO2. LPS (Escherichia coli serotype 026:B6, Sigma Chemical, St Louis, MO, USA) was dissolved (10 μg/ml) in saline solution. The PBMCs were cultured at a concentration of 1 × 107/ml per well of the 6-well plate, and were treated with LPS (the final concentration was 1 μg/ml) for 4, 8, 12 and 24 h. Cells were further centrifuged for 10 min at 3500 g and harvested for RNA extraction.
Ficoll hypaque
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Ficoll-Hypaque is a density gradient medium used for the separation and isolation of mononuclear cells from whole blood or other cell suspensions. It is a sterile, pyrogen-free solution composed of Ficoll, a high molecular weight, highly branched, hydrophilic polysaccharide, and sodium diatrizoate, a water-soluble compound of high density.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
Penicillin, by Thermo Fisher Scientific (5 mentions)
Rpmi 1640, by Thermo Fisher Scientific (5 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (4 mentions)
Hepes, by Thermo Fisher Scientific (3 mentions)
Human ab serum, by Cambrex (3 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (3 mentions)
Facscanto, by BD (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions)
Ficoll hypaque, by GE Healthcare (2 mentions)
Trypan blue, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Lps escherichia coli serotype 026 b6, by Merck Group (1 mentions)
Anti human cd14 pe, by BD (1 mentions)
24 well plate, by Corning (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Moflo cytometer, by Beckman Coulter (1 mentions)
48 protocols using ficoll hypaque
1
Isolating and Stimulating Porcine PBMCs
2
Isolation and culture of CD14+ monocytes
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Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of healthy donors by Ficoll-Hypaque (Gibco/BRL) density-gradient. CD14+ cells were separated by FACS sorting using anti-human CD14-PE (Pharmingen cat. 555398) in the MoFlo™ cytometer (Beckman Coulter). The monocytes obtained were on average 97% pure.
CD14+ monocytes at 106 cells/ml in 24-well plates (Costar, Cambridge, MA) were cultured in RPMI-1640 plus 5% human serum, 1 mM HEPES (GIBCO, cat. 15630), 2 mM L-Glutamine (GIBCO, cat. 25030), 100 UI/mL Penicillin (GIBCO, cat. 15070), 100μg/mL Streptomycin (GIBCO, cat. 15070), 50 μg/mL Gentamicin (GIBCO, cat. 15710) at 37°C in 5% CO2 atmosphere. Cultures were fed with fresh medium every 2 days for 6 days.
CD14+ monocytes at 106 cells/ml in 24-well plates (Costar, Cambridge, MA) were cultured in RPMI-1640 plus 5% human serum, 1 mM HEPES (GIBCO, cat. 15630), 2 mM L-Glutamine (GIBCO, cat. 25030), 100 UI/mL Penicillin (GIBCO, cat. 15070), 100μg/mL Streptomycin (GIBCO, cat. 15070), 50 μg/mL Gentamicin (GIBCO, cat. 15710) at 37°C in 5% CO2 atmosphere. Cultures were fed with fresh medium every 2 days for 6 days.
3
Isolation of Bone Marrow Mononuclear Cells
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BM aspirates were obtained from the remnant samples of 8 patients who underwent BM studies for diagnosis after acquiring informed consent. The protocol for this study was approved by the Institutional Review Board of Catholic University hospital of Daegu. Isolation of BM mononuclear cells (MNCs) was performed by following a previously described method [15 (link)]. Briefly, 4-6 mL of BM aspirate was mixed with 20 mL of Dulbecco's phosphate-buffered saline (DPBS, Gibco BRL Life technologies, Grand Island, NY). The mixture was resuspended and gently layered onto Ficoll-Hypaque (density, 1.073 g/mL; Gibco BRL Life technologies) and centrifuged at 900 g for 10 min at room temperature. The low-density MNC fraction was collected, combined with 25 mL DPBS, and centrifuged. Two samples were contaminated during cell culture and were therefore excluded from the study.
4
Isolation and Culture of Bone Marrow-Derived Cells
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Bone marrow aspirates (5 mL) were collected in the evacuated tubes containing heparin as an anticoagulant. The BMMNCs were separated from the bone marrow aspirates using 75% Ficoll–Hypaque suspended in RPMI-1640 (Gibco-BRL, Grand Island, NY, USA) without sterile fetal bovine serum (FBS) in a 75 mL culture bottle. The BMMNCs were then incubated for 4 h at 37°C under a saturated 5% CO2 atmosphere. The macrophages that adhered onto the bottle wall and the suspended non-adherent cells were discarded. The adherent cells were digested with 0.25% pancreatin containing 0.01% Ethylene Diamine Tetraacetie Acid (EDTA). The cells were cultured in LG-DMEM culture solution containing 10% FBS. The supernatant was removed and the cells were harvested using 0.2% trypsase solution. The BMMNCs were resuspended in RPMI-1640 and then stored for future use.
5
Allogeneic Stem Cell Transplant Monitoring
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All experiments were performed with authorization of the Institutional Review Board (IRB) for Human Research at the Catholic University of Korea. Study patients were the recipients of allogeneic SCT that were initially diagnosed with one of the hematologic diseases designated by the World Health Organization (WHO). Heparinized blood samples were collected from the recipients at a time of high CMV DNAemia for those diagnosed with CMV viremia (CMV+ group) and at any time after transplantation for those without CMV viremia (CMV- group). In addition, blood samples were collected from the recipients before conditioning (pre-HSCT group) and healthy donors (healthy donor group) before harvesting hematopoietic stem cells.
Mononuclear cells were isolated by overlaying the heparinized blood samples on a Ficoll-Hypaque gradient (density, 1.077; Lymphoprep; Gibco-BRL, Carlsbad, CA, USA), followed by centrifugation at 400×g for 30 minutes. The buffy coats were harvested and washed twice with phosphate-buffered saline (pH 7.4).
Mononuclear cells were isolated by overlaying the heparinized blood samples on a Ficoll-Hypaque gradient (density, 1.077; Lymphoprep; Gibco-BRL, Carlsbad, CA, USA), followed by centrifugation at 400×g for 30 minutes. The buffy coats were harvested and washed twice with phosphate-buffered saline (pH 7.4).
6
Activating Human and Mouse Lymphocytes
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Human peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by Ficoll-Hypaque gradient centrifugation, resuspended at 1×106/ml in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco) and activated by incubation with phytohemagglutinin (PHA, Sigma, St Louis, MO, USA) at 50 µg/ml for 36 h at 37°C. Lymphocytes from mouse spleens were prepared after lysing the erythrocytes with ammonium chloride and activated by incubation with concanavalin A (ConA,Sigma) at 5 µg/ml for 36 h at 37°C in 10% FBS RPMI 1640 medium. Expression of CD137 on the T cells was confirmed by flow cytometry (FACS Caliber, BD, San Jose, CA, USA) after double staining with FITC-conjugated anti-CD3 (OKT3, ebioscience, San Diego, CA, USA) and PE-conjugated anti-CD137(BD Biosciences, San Diego, CA, USA). RNA was extracted from activated and unstimulated human and mouse lymphocytes using TRIzol (Life Technologies, Carlsbad,CA, USA), and single-stranded cDNAs of IgG-Fc, CD137 and CD137L were synthesized using poly(dT) primers and M-MLV RT (Promega Corporation, Madison,WI, USA).
7
Isolation and Interaction of PBMC with Entamoeba
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Peripheral mononuclear blood cell (PMBC) was isolated from fresh human blood obtained in heparinized tubes from human volunteers. Whole blood was centrifuged by gradient of Ficoll-Hypaque (Gibco BRL); the PMBC was separated and washed three times with PBS and used immediately for assays of interaction with trophozoites of E. histolytica or E. dispar (1 : 6 ameba/lymphocytes).
8
Isolation and Cryopreservation of PBMCs
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Peripheral blood was obtained from 15 cancer patients (5 hepatocellular carcinoma, 8 lung cancer, and 2 colon cancer with liver metastasis) and 10 healthy subjects. All samples were processed at the National Cancer Institute in Naples under informed consent, as approved by the Institutional Review Board. Fresh human peripheral blood mononuclear cells (PBMCs), isolated by density gradient centrifugation using Ficoll-Hypaque, were cryopreserved at −150 °C in FBS (Gibco, Thermo Fisher Scientific) plus 10% DMSO until analysis.
9
Isolation and Characterization of Rat BMSCs
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BMSCs were isolated from the femurs and tibiae of male Wistar rats. After bone marrow cells were collected by flushing, nucleated cells were isolated with a density-gradient Ficoll-Hypaque (Gibco) and resuspended in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA) and 10% fetal bovine serum (FBS; Cultilab, Campinas, Brazil). Cells were incubated (37°C in 5% CO2) for 14 days as a primary culture. BMSCs were recovered by taking advantage of their tendency to adhere tightly to plastic, and the nonadherent cells were removed by washing. Flow cytometry analyses (FACS Canto; Becton Dickinson, East Rutherford, NJ, USA) were performed for CD31, CD44, CD90, CD45, and CD34 (Caltag Laboratories, Carlsbad, CA, USA), and we tested their potential for adipogenic and osteogenic differentiation, as previously described (data not shown) [2 (link), 11 (link)].
10
Mononuclear Cell Isolation and Benzene Exposure
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Mononuclear cells were isolated from bone marrow aspirations from healthy donors or patients with chronic benzene exposure using Ficoll‐hypaque and cultured in RPMI 1640 medium with 20% foetal bovine serum (both from Gibco, Grand Island, NY, USA). Also, BMMNCs had not been used for the following experiments until the percentage of CD38+ cells was more than 70% using flow cytometric analyses of the expression of CD34 and CD38. The CD34+ cells were isolated using a CD34 Microbead Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured in RPMI 1640 medium with 20% foetal bovine serum to a density of 1 × 106 cells/ml. The cells were treated with HQ (Jinshan chemical reagent factory, Zhejiang, China) at final concentrations of 5, 10, and 20 μmol/L for 12 hours. And the cells were also treated with C646 or Acetyl‐CoA (both from Sigma‐Aldrich, St.Louis, MO, USA).
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