Palatine tonsils were obtained at the Children’s Hospital of Columbia-Presbyterian Medical Center as residual material from three anonymous patients who had undergone elective tonsillectomy due to chronic tonsillitis in compliance with Regulatory Guideline 45 CFR 46.101 (b) (4 (link)) for Exempt Human Research Subjects of the U.S. Department of Health and Human Services and according to protocols approved by the Columbia University Institutional Ethics Committee. Tonsil specimens were placed on ice immediately after surgical removal. Mononuclear cells (MNC) were isolated by disaggregating tissues in RPMI 1640 medium (Gibco) followed by Ficoll-Isopaque (GE Healthcare) density centrifugation (27 (link)). MNC were stained using the following antibodies: anti-CD38-PE (clone HB7, BD Biosciences), anti-IgD-FITC (clone IA6-2, BD Biosciences), anti-CD3-FITC (clone UCHT1, Beckman Coulter), anti-CD184 (CXCR4)-Brilliant Violet 421 (clone 12G5, BioLegend), anti-CD83-APC (clone HB15e, BioLegend). Total GC (CD38+/IgD-/CD3-), DZ (CD38+/IgD-/CD3-/CXCR4high/CD83low) and LZ (CD38+/IgD-/CD3-/CXCR4low/CD83high) B cells were sorted using an Influx cell sorter (BD Biosciences). Data rendering was performed using FlowJo (TreeStar).
Ficoll isopaque
Manufactured by GE Healthcare
Sourced in United Kingdom
Ficoll-Isopaque is a density gradient medium used for the separation and isolation of various cell types, such as mononuclear cells, from blood or other biological samples. It facilitates the separation of cells based on their differing densities.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Anti cd3 fitc clone ucht1, by Beckman Coulter (2 mentions)
Anti igd fitc clone ia6 2, by BD (2 mentions)
Anti cd184 cxcr4 brilliant violet 421 clone 12g5, by BioLegend (2 mentions)
Anti cd83 apc clone hb15e, by BioLegend (2 mentions)
Anti cd38 pe clone hb7, by BD (2 mentions)
Influx cell sorter, by BD (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Facsaria flow cytometer, by BD (1 mentions)
Opteia cytokine elisa kits, by BD (1 mentions)
Citrate phosphate dextrose, by Merck Group (1 mentions)
Mtb h37ra, by American Type Culture Collection (1 mentions)
Lysotracker red, by Thermo Fisher Scientific (1 mentions)
Syto9, by Thermo Fisher Scientific (1 mentions)
Automacs pro separator, by Miltenyi Biotec (1 mentions)
5 aza 2 deoxycytidine aza, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Trypsin edta, by Lonza (1 mentions)
7 protocols using ficoll isopaque
1
Tonsil B Cell Subsets Isolation
2
Isolating Mononuclear Cells from JIA Synovial Fluid
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SF of JIA patients was incubated with hyaluronidase (Sigma-Aldrich) for 30 min at 37 °C to break down hyaluronic acid. SF mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll Isopaque density gradient centrifugation (GE Healthcare Bio-Sciences, AB) and were used after freezing in fetal calf serum (FCS) (Invitrogen) containing 10% DMSO (Sigma-Aldrich).
3
Tonsil Mononuclear Cell Isolation and Flow Cytometry
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Palatine tonsils were obtained at the Children’s Hospital of Columbia–Presbyterian Medical Center as residual material from anonymous patients who had undergone elective tonsillectomy due to chronic tonsillitis in compliance with Regulatory Guideline 45 CFR 46.101 (b)(4) for Exempt Human Research Subjects of the US Department of Health and Human Services and according to protocols approved by the Columbia University Institutional Ethics Committee. Tonsil specimens were placed on ice immediately after surgical removal. Mononuclear cells were isolated from three donors by disaggregating tissues in RPMI 1640 medium (Gibco), followed by Ficoll-Isopaque (GE Healthcare) density centrifugation (Klein et al., 2003 (link)). Tonsillar mononucleated cells were stained using the following antibodies: anti-CD38-PE (clone HB7; BD Biosciences), anti–IgD-FITC (clone IA6-2; BD Biosciences), anti–CD3-FITC (clone UCHT1; Beckman Coulter), anti-CD184 (CXCR4)–Brilliant Violet 421 (clone 12G5; BioLegend), and anti–CD83-APC (clone HB15e; BioLegend). Total GC (CD38+/IgD−/CD3−), DZ (CD38+/IgD−/CD3−/CXCR4high/CD83low), and LZ (CD38+/IgD−/CD3−/CXCR4low/CD83high) B cells were sorted using a FACSAria flow cytometer (BD Biosciences). Data rendering was performed using FlowJo (TreeStar).
4
Immunological Assays for Mycobacterium tuberculosis
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Mtb H37Ra was obtained from ATCC (ATCC no. 25177). RPMI 1640 medium, FITC-conjugated anti-human IgG antibody, Lysotracker Red, SYTO 9 and anti-rabbit Alexa Fluor antibody were from Invitrogen. Rabbit anti-iNOS and anti-LAMP-1 antibodies were from Thermo Pierce Biotech. Anti-CD14 mAb was from Boehringer-Mannheim GmbH. Rabbit anti-sheep RBC antibody was from Rockland Immunochemicals. mAbs against Mtb-Acr (IT-4) and LAM (CS-35) were gifted by UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. Affinity purified peroxidase-conjugated anti-human and anti-mouse IgG antibodies, anti-calmodulin antibody, o-phenylenediamine (OPD), diaminobenzidine (DAB), 2 µM amine-modified green fluorescent polystyrene beads, citrate phosphate dextrose and other laboratory chemicals were from Sigma-Aldrich. BD optEIA cytokine ELISA kits and Middlebrook (MB) media and supplements were obtained from BD Biosciences. Fluorescent NO probe FL2E was purchased from Strem Chemicals, Inc. Ficoll-isopaque was obtained from GE-Biosciences. Ninety-six-well clear glass bottom black imaging plates were from Thermo Nunc.
5
Isolation of CD14+ Monocytes from PBMC
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Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll‐isopaque (GE Healthcare) density gradient centrifugation. CD14+ monocytes were isolated by magnetic cell sorting using mouse anti‐human CD14 magnetic beads and autoMACS Pro Separator (Miltenyi Biotec; 130‐050‐201) according to manufacturer's instructions.
6
RNA Extraction and Analysis of Cell Lines
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Three cell lines, DAUDI, MCF-7 and A549, were cultured as previously described (25 (link),28 (link),29 (link)). All culture media were from Lonza (Verviers, Belgium). T cells were isolated from PBMC's by Ficoll-isopaque (GE Healthcare Life Sciences, Amersham, UK) gradient centrifugation and by positive magnetic selection (Miltenyi midiMacs, Miltenyi Biotech GmbH, Cologne, Germany) (21 (link)).
Total RNA was extracted from unstimulated DAUDI, MCF-7 and A549 cells, following routine passage using the RNeasy Mini Kit (QIAGEN, Venlo, Netherlands) according to the manufacturer's instructions. 1.5 × 107 DAUDI cells were seeded in a 75 cm3 flask. When they reached 70% confluence, they were stimulated with Interferon-γ (IFN-γ; 6 h, 5 ng/ml), Dexamethasone (Dex; 6 h, 100 nM) or 5-AZA-2′-deoxycytidine (AZA; 72 h, 10 μM) (Sigma-Aldrich, Diegem, Belgium). Cells were detached from the culture support using trypsin–EDTA (Lonza) and pelleted (5 min, 1671.6 × g). Subsequently, total RNA was isolated using the RNeasy Mini Kit (QIAGEN). RNA integrity was assessed using the Eukaryote Total RNA Nano assay with a RNA 6000 Nano chip on the Aligent 2100 Bioanalyzer (Aligent Technologies, Diegem, Belgium). The RNA quality assessment was based on the RNA integrity number (RIN). Only samples with a RIN value of >7 were used for further experiments.
Total RNA was extracted from unstimulated DAUDI, MCF-7 and A549 cells, following routine passage using the RNeasy Mini Kit (QIAGEN, Venlo, Netherlands) according to the manufacturer's instructions. 1.5 × 107 DAUDI cells were seeded in a 75 cm3 flask. When they reached 70% confluence, they were stimulated with Interferon-γ (IFN-γ; 6 h, 5 ng/ml), Dexamethasone (Dex; 6 h, 100 nM) or 5-AZA-2′-deoxycytidine (AZA; 72 h, 10 μM) (Sigma-Aldrich, Diegem, Belgium). Cells were detached from the culture support using trypsin–EDTA (Lonza) and pelleted (5 min, 1671.6 × g). Subsequently, total RNA was isolated using the RNeasy Mini Kit (QIAGEN). RNA integrity was assessed using the Eukaryote Total RNA Nano assay with a RNA 6000 Nano chip on the Aligent 2100 Bioanalyzer (Aligent Technologies, Diegem, Belgium). The RNA quality assessment was based on the RNA integrity number (RIN). Only samples with a RIN value of >7 were used for further experiments.
7
Isolating and Culturing Human PBLs
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Human peripheral blood leucocytes (PBLs) were isolated from 10 mL of peripheral blood from one healthy donor, under informed consent. Briefly, blood was diluted 1:1 with PBS layered over 10 mL Ficoll Isopaque (GE Healthcare) in a 50-mL tube and centrifuged for 40 minutes at 400
g. After centrifugation, the nucleated cells at the interface between Ficoll and supernatant were collected, washed two times with PBS, counted, and then maintained in RPMI medium with 10% FBS. Cells were then cultured in the presence or absence of the three different devices for 5 days.
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Human peripheral blood leucocytes (PBLs) were isolated from 10 mL of peripheral blood from one healthy donor, under informed consent. Briefly, blood was diluted 1:1 with PBS layered over 10 mL Ficoll Isopaque (GE Healthcare) in a 50-mL tube and centrifuged for 40 minutes at 400
g. After centrifugation, the nucleated cells at the interface between Ficoll and supernatant were collected, washed two times with PBS, counted, and then maintained in RPMI medium with 10% FBS. Cells were then cultured in the presence or absence of the three different devices for 5 days.
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