For single-cell suspensions, thymus and spleen were crushed and filtered through 80μm stainless steel mesh (Sefar Ltd., UK) in fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline (PBS) containing; 2% heat-inactivated fetal calf serum (FCS) (Life technologies); and 5 mM ethylenediaminetetraacetic acid (EDTA) (Life technologies)). For spleen, red blood cells were removed by gradient centrifiguation at 1600rpm for 25 min using 4 mL of Lymphocyte Separation Medium (Fischer). For IEL preparations, faecal material was flushed from lumen of small intestine with ice-cold PBS using a gavage needle. Fatty tissues, vasculature and Peyer’s patches were removed, followed by longitudinal opening and 60 min agitation in RPMI-1640 10% Newborn calf serum (NCS) (Life technologies) with 5 mM EDTA (Life technologies) at 37 °C. Cells were subsequently passed through an autoclaved column containing 0.7 g of nylon wool (Polysciences, USA), equilibrated with RPMI-1640 with 38 mM HEPES (Life technologies). IELs were enriched on a discontinuous Percoll gradient (40%/80% isotonic Percoll), before use and stained as described below.
Nylon wool
Manufactured by Polysciences
Sourced in United States
Nylon wool is a type of synthetic fiber material used in various laboratory applications. It is a durable, non-absorbent, and chemically inert substance that can be used for filtration, separation, and other purposes in a controlled scientific environment. The core function of nylon wool is to provide a reliable and consistent material for specialized lab equipment and procedures.
Automatically generated - may contain errors
Lab products found in correlation
Ethylenediaminetetraacetic acid, by Thermo Fisher Scientific (1 mentions)
Lymphocyte separation medium, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Newborn calf serum ncs, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Ack lysing buffer, by Lonza (1 mentions)
70 m cell strainer, by BD (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Cmf hbss, by GE Healthcare (1 mentions)
Complete rpmi 1640, by GE Healthcare (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Gentamycin, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Cfse labeling kits, by Thermo Fisher Scientific (1 mentions)
6 protocols using nylon wool
1
Isolation of Lymphocyte Subsets
2
Immunomodulatory effects of APS on T and B cells
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The immunomodulation properties of APS were evaluated by determining their effects on proliferation and cytokine expression in cultured T and B lymphocytes [33 (link)]. Briefly, the spleen lymphocytes were isolated from RAW 264.7 mouse, which were free of red blood cells from treatment with a lysis buffer (0.15 M NH4Cl, 0.01 M KHCO3, and 0.1 mM Na2EDTA, pH 7.4). To remove adherent cells, such as macrophages, the spleen cells were incubated for 1 hour in Petri dishes at 5 × 106 cells/mL. The B and T cells were separated using the nylon wool method (Polysciences Inc., Warrington, PA, USA) according to the manufacturer’s instructions [34 (link)]. After washing with PBS buffer solution and counting with a counter board, the isolated B and T lymphocytes were used for subsequent assays.
3
Isolation of Enriched NK/LAK Cells
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NK/LAK cells were generated from splenocytes of naive mice. A 10-mL syringe was filled with 1.2 g of Nylon Wool (Polysciences) and sterilized. Five spleens were collected from 6–8 weeks old naive female C57Bl/6 WT mice and single-cell suspensions were made by passage over a 100 μm cell strainer. Ery-lysis was performed using ACK-buffer and stopped with complete medium. The Nylon Wool “column” was prepared using IMDM, containing 5% FCS, 1% AA, 2 mM Glutamine and 0.05% β-mercaptoethanol (medium) for 1 h at RT. Splenocytes were passaged over a Nylon Wool column and were plated at a density of 2.5 × 106 cells/mL in medium containing 1000 IU/mL of human rIL-2. On day 3 and 6, non-adherent cells were washed away by extensive medium washings. On day 7, adherent cells were harvested using 1.5 mM EDTA. The harvested adherent cells were >90% NK1.1+NKp46+ cells, as determined by flow cytometry.
4
Isolation and Culture of Mouse T Cells
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Isolated spleen from DBA/1J mice was disaggregated in RPMI-1640 medium. Splenocytes were filtered with a 70 µm cell strainer (BD Biosciences) and then the red blood cells were lysed using ACK Lysing Buffer (Lonza, Basel, Switzerland). T cells were enriched by passing the splenocytes through a nylon wool (Polysciences, Inc., Warrington, PA, USA) column. DCs and T cells were cultured directly in the same plates. Enriched T cells (1 × 106 cells/mL) were employed as responders, and T74-DCs or NT-DCs (1 × 105 cells/mL) were stimulators. T Cells and DCs were incubated at 37 °C for 72 h in RPMI-1640 medium containing 10% FBS.
5
Isolation and Culture of Chicken Intestinal and Splenic Immune Cells
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Chicken intestinal epithelial lymphocytes (IELs) and splenic mononuclear cells (SMCs) were isolated from jejunum and spleen, respectively, as previously described with modification [6 (link),14 (link)]. Briefly, jejunum was collected aseptically from healthy or E. necatrix-infected chicken and washed with ice-cold Ca2+- and Mg2+-free HBSS (CMF-HBSS, GE Healthcare, Pittsburgh, Pennsylvania, USA) containing 1 mM dithiothreitol (MilliporeSigma, St. Louis, Missouri, USA) followed by incubation with CMF-HBSS containing 0.5 mM EDTA and 5% FBS (GE Healthcare) for 20 min at 37°C with constant swirling. Cells released into the supernatant were pooled, passed through nylon wool (Poly Sciences, Warrington, Pennsylvania, USA), and purified by a Histopaque-1077(MilliporeSigma) density gradient method. For SMCs, collected spleen was homogenized using gentleMACS Dissociator (Miltenyi Biotec, Gaithersburg, USA) and purified by the same method described above. Freshly purified lymphocytes were cultured in complete RPMI-1640 (GE Healthcare) supplemented with 10% FBS, penicillin/streptomycin (10,000 unit/ml, Invitrogen, Carlsbad, California, USA), 50 μg/ml gentamycin, 25 mM HEPES, and 55 μM 2-Mercaptoethanol (all from MilliporeSigma). The chicken macrophage cell line HD11 was maintained in complete DMEM supplemented the same as RPMI-1640 described above.
6
T Cell Proliferation Assay with DCs
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Splenocytes were isolated from wild type C57BL/6 mice spleen and disaggregated in RPMI 1640 medium. CD3+ T cells were isolated by passing the splenocytes through a nylon wool (Polysciences Inc., Warrington, PA, USA) column. Nylon-wool column-purified T cells from spleens were labeled using CFSE labeling kits (Molecular Probes, Oregon, USA) according to the manufacturer’s instructions. Cells were then co-cultured with DCs at a DC:T cell ratio of 1:10. CFSE-labeled purified CD3+ T cells (1 × 106 cells/ml) were used as responders and imDCs or mDCs (1 × 106 cells/ml) were used as stimulators. Cells were co-cultured at 37 °C for 72 h in 2 ml of RPMI 1640 supplemented with 10% FBS.
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