Blood was collected in heparinized tubes after which PBMCs were isolated by performing a Ficoll-Paque™ PLUS gradient (GE Healthcare, UK) within 4 h after the blood draw. PBMCs were harvested and the monocyte population was enriched via positive selection with CD14-coupled magnetic micro beads according to the manufacturers protocol (Miltenyi Biotec GmbH, Germany). The CD14+ cells were suspended in culture medium (Gibco® RPMI 1640 substituted with 100 Units/mL penicilin, 100 μg/mL streptomycin and 2 mM L-glutamin (BioWhittaker, Belgium)), collected in cryovials, slowly frozen in freezing medium (50% culture medium, 40% fetal bovine serum and 10% dimethylsulfoxide) and after one night at −80 °C, stored in liquid nitrogen until further use. To generate mo-MΦs, monocytes were thawed, immediately washed and resuspended in culture medium supplemented with 10% human serum (Sanquin, The Netherlands), and plated (1.25*10^5 cells/well) in 48-well Corning® Costar® cell culture plates (Corning, NY) for seven days to generate mo-MΦs.35 The cells were then used for qPCR analysis or pro/anti-inflammatory stimulation assays.
Ficoll paque plus gradient
Manufactured by GE Healthcare
Sourced in United States, Sweden, United Kingdom, Japan, Switzerland, Australia
Ficoll-Paque PLUS gradient is a ready-to-use medium for the isolation of mononuclear cells from blood or bone marrow. It is designed for the separation of different cell types by density gradient centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Rpmi 1640, by Thermo Fisher Scientific (5 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Easysep human cd4 t cell enrichment kit, by STEMCELL (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (2 mentions)
Easysep human na ve cd4 t cell isolation kit, by STEMCELL (2 mentions)
Easysep human cd25 positive selection kit, by STEMCELL (2 mentions)
Dif quik, by Merck Group (2 mentions)
Dynal cd4 positive isolation kit, by Thermo Fisher Scientific (2 mentions)
Anti cd3 and anti cd28 coated beads, by Thermo Fisher Scientific (2 mentions)
Interleukin 2 (il 2), by Merck Group (2 mentions)
Facsdiva software, by BD (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Facsaria cell sorter, by BD (2 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (2 mentions)
Facscalibur, by BD (2 mentions)
Facsaria, by BD (2 mentions)
71 protocols using ficoll paque plus gradient
1
Isolation and Differentiation of Human Monocyte-Derived Macrophages
2
Isolation and Characterization of Human B Cells
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Blood was obtained in accordance with the policies established by the National Yang-Ming University Institutional Review Board. Blood was withdrawn from healthy donors, and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque PLUS gradient centrifugation (GE Healthcare, Chicago, IL). B cells were positively isolated from PBMCs using CD19 microbeads (Miltenyi Biotec, Auburn, CA). Cell purity was determined using anti-human CD20-APC (BioLegend, San Diego, CA) staining. B cell purity using CD19 microbeads purification was > 95% in all experiments. Cells were cultured in RPMI 1640 medium containing 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and 10% fetal calf serum (Life Technologies, Grand Island, NY). CTLA-4-Ig and L6-Ig (control-Ig) for use in the in vitro assays were provided by Bristol-Myers Squibb. L6-Ig is a chimeric fusion protein consisting of the V region of the murine L6 antigen and the human IgG1 Fc portion, and it was used as a control in our studies.
3
Splenocyte and Monocyte Isolation
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The spleens were removed and pressed through a nylon cell strainer (BD Falcon, Tewksbury, MA). The effluent samples were further filtered, suspended in red blood cell lysis buffer (eBioscience, San Diego, CA), and centrifuged. The splenocytes were checked for viability and used for Western blot or real-time RT-PCR analyses. Peripheral blood mononuclear cells (monocytes) were isolated with a Ficoll-Paque PLUS gradient (GE Healthcare, Pittsburgh, PA).
4
Evaluation of GM-CSF-induced STAT5 Phosphorylation
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GM-CSF-stimulated phosphorylation of STAT5 in peripheral blood monocytes (PBMC) and granulocytes was evaluated in six patients according to a modified protocol by Suzuki et al. [11 (link)]. Heparinized blood was processed within 24 hours. Mononuclear cells and granulocytes were isolated using a Ficoll-Paque plus gradient (GE Healthcare, Uppsala, Sweden) and Leucosep tubes (Greiner Bio One, Solingen, Germany). Cell suspensions were incubated with or without GM-CSF (Leukine, Berlex, Bayer Healthcare, Seattle, WA; 100 ng/ml, 15 minutes, 37°C). Expression of phosphorylated STAT5 (pSTAT5) was measured by flow cytometry (BD FACSCanto). In a subset of cases, cell lysates were also evaluated by Western blotting using anti-STAT5 (Santa Cruz Biotechnology; Santa Cruz, CA), anti-phospho-STAT5 (Cell Signaling), or anti-beta actin (Santa Cruz) as primary antibodies.
5
PBMC Isolation and IL-15 Stimulation
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Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation using a Ficoll-Paque Plus gradient (GE Healthcare, Cat #17-1440-02), then washed by centrifugation, and resuspended in RPMI 1640 media (Gibco) supplemented with 10% FBS (Gibco) and 100 U/ml penicillin, and 100 μ g/ml streptomycin (Gibco). The PBMCs were adjusted to a concentration of 1 × 106 cells/mL in 500 mL of RPMI 1640 media. Culturing was performed in the presence or absence of recombinant IL-15 (10 ng/mL) (Petrotech, Cat. #200-01B) and incubated for 18h at 37°C under 5% CO2 for 18h.
6
Isolation and Characterization of Immune Cells
7
Isolation and Activation of CD4+ T Cells
8
Exosome-induced PBMC Cytokine Response
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Peripheral blood mononuclear cells (PBMCs) were isolated from heparinzed peripheral venous blood of HCs by density gradient centrifugation using Ficoll-paque plus gradient (GE Healthcare Biosciences, Uppsala, Sweden). Cell viability was assessed with trypan blue dye exclusion. PBMCs (5 × 106 cells/mL) were stimulated with 10 μL purified exosomes in RPMI-1640 supplemented with 100 U/mL penicillin and 100 μL/mL streptomycin for 24 h at 37 °C in a 5 % CO2 incubator.
To investigate the involvement of TLRs, PBMCs were pretreated with TLR1/2 antagonist (CU-CPT22; Calbiochem, MA, USA), TLR4 antagonist (LPS-RS ultrapure; Invivogen, CA, USA), TLR7 antagonist (ODN20958; Invivogen, CA, USA), or TLR9 antagonist (ODN2088; Invivogen, CA, USA) for 1 h. Then the cells were incubated with exosomes for 24 h. Following stimulation, levels of IFN-α, TNF-α, IL-1β, and IL-6 in the supernatants were measured using ELISA according to the manufacturers’ instructions (IFN-α, PBL Assay Science, NJ, USA; TNF-α, IL-1β, and IL-6, BD Biosciences, CA, USA).
To investigate the involvement of TLRs, PBMCs were pretreated with TLR1/2 antagonist (CU-CPT22; Calbiochem, MA, USA), TLR4 antagonist (LPS-RS ultrapure; Invivogen, CA, USA), TLR7 antagonist (ODN20958; Invivogen, CA, USA), or TLR9 antagonist (ODN2088; Invivogen, CA, USA) for 1 h. Then the cells were incubated with exosomes for 24 h. Following stimulation, levels of IFN-α, TNF-α, IL-1β, and IL-6 in the supernatants were measured using ELISA according to the manufacturers’ instructions (IFN-α, PBL Assay Science, NJ, USA; TNF-α, IL-1β, and IL-6, BD Biosciences, CA, USA).
9
Isolation and Activation of CD4+ T Cells
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CD4+ T cells were extracted from healthy donor blood (Etablissement Francais du Sang, EFS) with continuous-flow centrifugation leukophoresis product using density centrifugation on a Ficoll-Paque Plus gradient (GE HealthCare Life Science). CD4+ lymphocytes were purified from peripheral blood mononuclear cells (PBMCs) by negative selection with CD4+ T Cell isolation kit (Miltenyi Biotec) and activated with an equal amount of the provided biotinylated antibodies against CD2, CD3 and CD28 loaded on MACSiBeads Particles (Beads-to-cell ratio 1:2) (T cell Activation/Expansion kit; Miltenyi Biotec) for 3 days. CD4+ lymphocytes were cultured in RPMI medium 1640 (1X) + GlutaMAX (Gibco, Life Technologies) supplemented with 10% FBS (Gibco, Life Technologies), penicillin (100 U/ml), streptomycin (100 µg/ml), amphotericin B (0.25 µg/ml) (Gibco, Life Technologies), and stimulated with 30 U/ml IL-2 (Sigma Aldrich) (37 °C, 5% CO2). Activated cells were characterized by flow cytometry with CD25-APC (BD Pharmingen), CD69-PE (BD Pharmingen) HLA-DR-PERCP-Cy5.5 (BD Bioscience) and KI-67-FITC (DAKO).
HeLa and HEK293T cells were cultivated at 5% CO2 and 37 °C in DMEM (Life technologies) supplemented with 10% FBS (Gibco, Life technologies).
HeLa and HEK293T cells were cultivated at 5% CO2 and 37 °C in DMEM (Life technologies) supplemented with 10% FBS (Gibco, Life technologies).
10
Isolation of Human PBMCs for Phagocytosis
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Human peripheral blood mononuclear cells (PBMC) were isolated from the peripheral venous blood of healthy volunteers. Briefly, whole blood (20 mL) was mixed with 7 mL of a 6% dextran solution and 15 mL of HBSS and allowed to stand for 30 minutes at 25 °C until stratification occurred. The upper leukocyte-rich plasma layer was transferred to a new tube containing endotoxin-free Ficoll-Paque PLUS gradient (GE Healthcare Japan, Tokyo, Japan) and was centrifuged (500 g, 30 min, 25 °C). The cell layer was harvested and subsequently, the cells were resuspended at 100 million PBMC per mL in Monocyte Attachment Medium (Promocell, Heidelberg, Germany). The cells were cultured for 10 days as per the manufacturer’s protocol and used for the phagocytosis assay, as described above. The protocol was approved by the Ethical Review Committee at the Teikyo University School of Medicine (no. 15-192-2). All participants gave a written informed consent prior to their inclusion in the study.
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