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23 protocols using Ficoll-Hypaque

Heparinized blood (5 ml) was 2-fold diluted with PBS, and subjected to Ficoll-Hypaque (Axis-Shield PoC AS, Norway) density gradient centrifugation at 800 × g for 20 min. The PBMC layer was collected, washed twice with PBS, and resuspended with ice cold PBS containing 0.1% NaN3 at 1 × 106 cells/ml. Cells were incubated with PE-conjugated anti-feline CD4 mAb and FITC-conjugated anti-feline CD8 mAb at 4 °C for 30 min. After washing, the cells were stored in fluorescence buffer prior to analysis on a flow cytometer (Cytomics FC500, Beckman Coulter, U.S.A.). The small lymphocyte and lymphoblast populations were gated on the basis of the cell size and granularity (forward and side scatter). For each sample 100,000 events were recorded, and the percentage of CD4+ and CD8+ T lymphocyte population was calculated. Absolute CD4+ and CD8+ T lymphocyte counts were determined from complete blood count, differential cell counts and percentage of CD4+ and CD8+ T lymphocyte population.
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PBMCs were isolated from peripheral blood by Ficoll-Hypaque density gradient centrifugation (Lymphoprep; Axis-Shield, Oslo, Norway) and rested overnight in RPMI-1640 medium supplemented with 25 mM HEPES, 2 mM l-glutamine (all from Thermo Fisher Scientific, Waltham, MA, USA), 10% FBS (Sigma-Aldrich, St. Louis, MO, USA), 50 μg/ml gentamicin (Thermo Fisher Scientific), and 100 μg/ml Normocin (InvivoGen, San Diego, CA, USA) (complete medium). Vα7.2+ cells were isolated from PBMCs with anti-Vα7.2 PE- or APC-conjugated mAb (BioLegend, San Diego, CA, USA), followed by positive selection with MACS anti-PE or anti-APC microbeads, respectively (Miltenyi Biotec, San Diego, CA, USA), according to manufacturer’s instructions. Monocytes were obtained from peripheral blood by negative selection with the RosetteSep human monocyte enrichment cocktail (StemCell Technologies), according to the manufacturer’s instructions.The E. coli strain D21 was cultured overnight to late stationary phase at 37°C in Luria-Bertani broth. Live bacteria were counted by the standard plate-counting method, and counts were expressed as CFU per milliliter. Live E. coli was divided in aliquots in 50% glycerol/50% FCS and stored at −80°C until needed for functional assays.
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Peripheral blood was collected in heparinized tubes from 28 healthy volunteers (15 men and 13 women, with ages ranging from 28 to 53). All donors provided written informed consent prior to study participation. To investigate clinical applicability, we collected blood samples from patients with liver diseases [n = 26; HCC (n = 19) and liver cirrhosis (n =7)] to evaluate NK cell activity utilizing the overnight WB NK cytotoxicity assay. This study was approved by the Institutional Review Board of the Samsung Medical Center, Seoul, Korea (IRB No. SMC 2018-11-005-004). Peripheral blood was used for determining the complete blood count (CBC), and for performing the CD107a degranulation assay and the NK cytotoxicity assay using WB and PBMCs. The CBC was measured on a Sysmex XE-2100 analyzer (Sysmex, Kobe, Japan). Human PBMCs were isolated from healthy adult donors using density-gradient centrifugation with Ficoll-Hypaque (d = 1.077, LymphoprepTM; Axis-Shield, Oslo, Norway) and washed twice with phosphate-buffered saline (PBS) (Welgene, Gyeongsan-si, Gyeongsangbuk-do, Korea).
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PBMC were obtained from peripheral blood by density gradient separation with Ficoll-Hypaque (Axis-Shield, Oslo, Norway). CD45RA+ CD4+ T cells and CD27- B cells were isolated from PBMC using antibody-coated magnetic Microbeads (MACS, MiltenyiBiotec Inc, CA, USA), according to the manufacturer’s protocol. Separation was assessed by flow cytometry (purity 90% and 97%, respectively).
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Plural fluid cells samples were isolated by Ficoll-Hypaque (Axis-Shield) density gradient centrifugation and were suspended to a final concentration of 1 × 106 cells/mL in complete RPMI 1640 medium (Gibco). CD4+ T cells were purified from 1 × 107 of PFCs in 40 μL sorting buffer using 10 μL of CD4+ T-cell Negative Sorting Microbeads (Miltenyi Biotec). When required, 2 μL CD3 and 2 μL CD4 goat polyclonal antibodies (Santa Cruz Biotechnology) were used to block the action site before sorting by E7/HLA-DR tetramers in total 50 μL sorting solution. Then, MTB peptide or non-peptide/HLA-DR tetramers bound CD4+ T cells were purified from CD4+ T cells using final concentration 6 μg/mL of MTB peptide or non-peptide/HLA-DR tetramers labeled with PE prepared before and Anti-PE MicroBeads (Miltenyi Biotec, 10 μL added in 90 μL sorting solution). Meanwhile, the cells that were not in combination with Anti-PE magnetic beads namely unbound CD4+ T cells were also preserved.
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Bone marrow (BM) cells were flushed from mouse femurs and tibiae and prepared as single-cell suspension after lysing the red blood cells. Peripheral blood mononuclear cells (PBMC) in mouse blood samples were isolated with Ficoll-Hypaque (Axis-Shield). All cells labeled with antibodies were immediately analyzed on a FACS Calibur (BD Biosciences, Mountain View, CA, USA).
For the analysis of MDSCs, the appropriate number of cells was pre-incubated with optimal concentration surface marker antibodies (eBioscience), anti-mouse CD11b-APC, and anti-mouse Gr1-PE.
For the analysis of PMN-MDSCs and M-MDSCs, the appropriate number of cells was pre-incubated with optimal concentration surface marker antibodies (eBioscience), anti-mouse CD11b-APC, anti-mouse Ly6G-FITC, and anti-mouse Ly6C-PE.
For the analysis of immature or undifferentiated markers CD11c, F4/80, CD80, and MHCII on MDSCs, the appropriate number of cells was pre-incubated with optimal concentration surface marker antibodies (eBioscience), anti-mouse CD11b-APC, anti-mouse Gr1-PE or anti-mouse Gr1-FITC, and anti-mouse CD11c-FITC, anti-mouse F4/80-FITC, anti-mouse CD80-PE, or anti-mouse MHCII-PE.
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The blood of each child was collected in the morning after 1–2 days of hospitalization, and 3 mL of blood was collected in a heparin-anticoagulation tube. Blood samples were placed in a 4–6°C refrigerator and were analyzed within 6 h. Peripheral-blood mononuclear cells (PBMCs) were isolated from heparinized blood using the Ficoll-Hypaque (Axis-Shield, Oslo, Norway) density-gradient centrifugation method. Freshly isolated heparin-anticoagulated blood diluted in phosphate-buffered saline (PBS, 1:1) was layered on the surface of Ficoll at a ratio of 2:1 and was centrifuged at 1,600 g for 20 min at 20°C, with no breaks. The cells were harvested from the Ficoll interface.
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Human peripheral blood mononuclear cells (PBMCs) were separated from the peripheral blood of healthy donors by gradient centrifugation on Ficoll-Hypaque (Lymphoprep, AXIS-SHIELD PoCAs, Oslo, Norway) at room temperature (RT). The concentration of isolated PBMC was adjusted to 2 × 106 cells/mL in RPMI 1640 including 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (all from EuroClone Spa, Milano, Italy). Twenty hours later, PBMCs were washed, and used for experiments described below.
PBMCs in the majority of experiments were activated with 5 µg/mL phytohemoagglutinin (PHA) for 24 h prior to and during the phase of EV exposure. A volume corresponding to 8µg of proteins of EVs was added to the PBMC suspension (2 × 105 cells/200µL) for 4 days.
PBMCs were stained with the following kits: Muse™ Human CD8 T Cell Kit and Muse™ Human CD4 T Cell Kit (MIM100102 and MIM100101, Merck Millipore, Billerica, MA, USA). A minimum of 10,000 cells per sample was acquired and analyzed using Muse™ Cell Analyzer.
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Patient-derived AML cells samples were obtained with informed consent as part of a clinical protocol approved by the Institutional Review Board of The University of Texas, M.D. Anderson Cancer Center. Mononuclear cells were purified by Ficoll Hypaque (Axis Shield, Oslo, Norway) density centrifugation following the manufacturer’s protocol. Mononuclear cells were washed once with sterile 1× PBS then suspended in complete RPMI media containing 20% FBS. Cells were counted to determine the number of cells isolated prior to immuno-magnetic selection. CD34 + AML blast progenitor cells were purified by immuno-magnetic beads conjugated with anti-CD34 antibody following the manufacturer’s protocol (StemCell Technologies, Vancouver, British Columbia) prior to utilization in the cell viability assays, RNA expression, and immunoblot analyses.
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Patient-derived AML cells samples were obtained with informed consent as part of a clinical protocol approved by the Institutional Review Board of The University of Texas, M.D. Anderson Cancer Center. Mononuclear cells were purified by Ficoll Hypaque (Axis Shield, Oslo, Norway) density centrifugation following the manufacturer’s protocol. Mononuclear cells were washed once with sterile 1× PBS then suspended in complete RPMI media containing 20% FBS.
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