Nylon wool column

Manufactured by Polysciences

Sourced in United States

The Nylon Wool Column is a laboratory equipment used for the purification and separation of biomolecules. It consists of a packed column made of nylon fibers that can be used for the adsorption and elution of target molecules from complex mixtures.

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Lab products found in correlation

Microplate reader, by Bio-Rad (2 mentions) Magnetic activated cell sorter system, by Miltenyi Biotec (1 mentions) Tissue culture plate, by Corning (1 mentions) Bdm 2 3 butanedione monoxime, by Merck Group (1 mentions) Human il 2, by R&D Systems (1 mentions) Phoenix eco packaging cells, by American Type Culture Collection (1 mentions) Cd3 cd28 dynabead, by Thermo Fisher Scientific (1 mentions) Lymphoprep, by Merck Group (1 mentions) Rpmi 1640 medium, by Corning (1 mentions) Six well plate, by Corning (1 mentions) Scintillation counter, by PerkinElmer (1 mentions) Ficoll gradient separation, by GE Healthcare (1 mentions) 70 μm cell strainer, by BD (1 mentions)

Close spelling variants of this product

Nylon wool fiber columns Nylon wool

11 protocols using nylon wool column

Purification and Activation of CD4+ T Cells

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CD4⁺ T cells were purified by the magnetic activated cell sorter system (Miltenyl Biotec Inc., Sunnyvale, CA) as described previously [27 (link), 28 (link)]. Briefly, cells were incubated in a nylon wool column (Polysciences Inc., Warrington, PA) to remove B cells and macrophages. Enriched T cell populations were then incubated with biotinylated anti-CD4 (GK 1.5) mAb followed by streptavidin-conjugated microbeads and passed through a magnetized column. The purified T cell fractions were > 97% CD4⁺ and were > 99% viable. Cells were resuspended in complete medium and purified CD4⁺ T cells (4 x 106 cells/ml) were cultured with or without 1 mg/ml of OVA in the presence of T cell-depleted, irradiated (3000 Rads) splenic Ag-presenting cells (APCs). These APCs were derived from naïve mice and were placed in 96-well or 24-well tissue culture plates (Corning Glass Works, Corning, NY) for 5 days at 37°C in a moist atmosphere of 5% CO2 in air. In some experiments, culture supernatants were harvested after 2 or 5 days of incubation and were then subjected to a cytokine-specific ELISA.

Aso K., Tsuruhara A., Takagaki K., Oki K., Ota M., Nose Y., Tanemura H., Urushihata N., Sasanuma J., Sano M., Hirano A., Aso R., McGhee J.R, & Fujihashi K. (2016). Adipose-Derived Mesenchymal Stem Cells Restore Impaired Mucosal Immune Responses in Aged Mice. PLoS ONE, 11(2), e0148185.

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Isolation and Culture of Immune Cells and Cardiomyocytes

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Macrophages were prepared by differentiating bone marrow cells from Mcpt4^−/− and WT mice in macrophage colony-stimulating factor (M-CSF) (20 ng/ml, Cat# 416-ML-050, R&D Systems) for 10 days. Total T cells were separated from splenocytes using a nylon-wool column (Cat# 21759, Polysciences, Inc., Warrington, PA) to yield a crude T cell preparation. CD4⁺ and CD8⁺ T cells were prepared as described previously [28 (link)]. Macrophages, CD4⁺ and CD8⁺ T cells were cultured in RPMI containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.
Adult cardiomyocytes from Mcpt4^−/− and WT mice were isolated and cultured as previously reported [29 ,30 (link)]. After centrifugation at 20 g for 5 minutes, cardiac fibroblasts in the supernatant were collected and cultured in high-glucose DMEM medium (Cat# 10566016, Invitrogen, Carlsbad, CA) containing 10% FBS and 1% penicillin/streptomycin. And the remaining pellet of cardiomyocytes was re-suspended in MEM containing 1% BSA, 1% penicillin/streptomycin and 10 mM 2,3-Butanedione monoxime (BDM) (Cat# B0753, Sigma-Aldrich). The medium was changed every 48 hours.

Wang Y., Liu C.L., Fang W., Zhang X., Yang C., Li J., Liu J., Sukhova G.K., Gurish M.F., Libby P., Shi G.P, & Zhang J. (2019). Deficiency of mouse mast cell protease 4 mitigates cardiac dysfunctions in mice after myocardium infarction. Biochimica et biophysica acta. Molecular basis of disease, 1865(6), 1170-1181.

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Mouse CAR-T Cell Production Protocol

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Mouse CD19scFv-CD28-CD3ζ CAR (m1928z) construct with GFP in SFG retroviral vector was described before and provided by M. Davila at the Moffitt Cancer Center³⁴. The isolation, activation and transduction of mouse T cells followed the procedure described before^{34 ,35}. Briefly, the spleens were collected from female C57Bl/6 mice and T cells were enriched from splenocytes by passage over a nylon wool column (Polysciences). Mouse T cells were then activated with CD3/CD28 Dynabeads (Thermo Fisher) following the manufacturer’s instructions and cultured in the presence of human IL-2 at 30 IU ml⁻¹ (R&D Systems). Retrovirus was produced by transfecting Phoenix-Eco packaging cells (ATCC) and spinoculations were done twice with retroviral supernatant. mCART19 cells were expanded for 10–14 days as described³⁵. UT-T were produced following the same procedure without viral transduction.

Staedtke V., Bai R.Y., Kim K., Darvas M., Davila M.L., Riggins G.J., Rothman P.B., Papadopoulos N., Kinzler K.W., Vogelstein B, & Zhou S. (2018). Disruption of a self-amplifying catecholamine loop reduces cytokine release syndrome. Nature, 564(7735), 273-277.

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Induction of Experimental Autoimmune Uveitis

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The method used to induce tEAU has been reported previously (4 (link)). Briefly, T cells from mice immunized 12 days previously with peptide IRBP_1–20 were purified from draining lymph nodes and spleen cells by passage through a nylon wool column (Polysciences, Warrington, PA, USA), then 1 × 10⁷ cells in 2 ml of RPMI 1640 medium were added to each well of a six-well plate (Costar, Corning, NY, USA) and stimulated with 20 µg/ml of IRBP_1–20 in the presence of 1 × 10⁷-irradiated syngeneic spleen cells as antigen-presenting cells (APCs). After 2 days, activated lymph blasts were isolated by gradient centrifugation on Lymphoprep (Sigma-Aldrich) and injected intraperitoneally (i.p.) in 0.2 ml of PBS into naive B6 recipients (5 × 10⁶ cells/mouse). The clinical course of the disease was assessed by indirect fundoscopy once or twice a week and graded as described previously (13 (link)).

Yun J., Jiang G., Wang Y., Xiao T., Zhao Y., Sun D., Kaplan H.J, & Shao H. (2017). The HMGB1–CXCL12 Complex Promotes Inflammatory Cell Infiltration in Uveitogenic T Cell-Induced Chronic Experimental Autoimmune Uveitis. Frontiers in Immunology, 8, 142.

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Allogeneic T Cell Proliferation Assay

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Allogeneic spleen-derived T cells were separated by a nylon wool column (Polysciences, PA, USA) according to the manufacturer’s protocols. Then, imDCs, mDCs and Met-mDCs were separately cocultured with T cells (DCs: T cells = 1:1, 1:10, 1:50 and 1:100) in a 37 °C incubator with 5% CO₂ for 48 h. Then, the cocultured cell suspensions were collected and fully mixed. Then, 100 μl of these mixtures was seeded in a 96-well plate, and 10 μl of CCK-8 solution was added. After 4 h, T-cell proliferation was quantified by measuring the optical density at 450 nm with a microplate reader (Bio-Rad, Hercules, CA, USA). To analyze T_reg differentiation, the cocultured cells in each group (1:10 mix ratio) were labeled with FITC-conjugated CD4, APC-conjugated CD25 and PE-conjugated Foxp3 Abs and then measured by flow cytometry. Triple-positive cells were identified and analyzed by flow cytometry.

Liu X., Yu P., Xu Y., Wang Y., Chen J., Tang F., Hu Z., Zhou J., Liu L., Qiu W., Ye Y., Jia Y., Yao W., Long J, & Zeng Z. (2023). Metformin induces tolerogenicity of dendritic cells by promoting metabolic reprogramming. Cellular and Molecular Life Sciences, 80(10), 283.

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Allogeneic T Cell Proliferation Assay

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Treg Suppression of Effector T Cells

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CD8+CD122+PD-1+ Tregs from naive B6 mice were first isolated by FACS sorting. They were then cultured with B6-derived T cells (Teff), which were enriched via nylon wool columns (Polysciences, Warrington, PA), in 96-well plates in the complete RPMI 1640 medium (10%FCS, 2mM glutamine, 100U/ml penicillin, and 100μg/ml streptomycin). The ratios of Treg to Teff were 1:4 (Treg: 1×10⁵/well and Teff: 4×10⁵/well). Irradiated BALB/c spleen cells (2.5×10⁵/well) were added to the culture to serve as donor-derived stimulators, as described previously [33 (link), 34 (link)]. Three and five days later, cells were harvested and analyzed by a Scintillation counter (PerkinElmer, Meriden, CT). Cells were pulsed with [³H]-Thymidine for the last 8 hours before analysis.

Liu H., Wang Y., Zeng Q., Zeng Y.Q., Liang C.L., Qiu F., Nie H, & Dai Z. (2017). Suppression of allograft rejection by CD8+CD122+PD-1+ Tregs is dictated by their Fas ligand-initiated killing of effector T cells versus Fas-mediated own apoptosis. Oncotarget, 8(15), 24187-24195.

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T Cell Purification from PBMC

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T cells were purified from PBMC of control donors and CLL patients as previously described [15 (link)]. Briefly, PBMC were washed three times with PBS and passed through nylon wool columns (Polysciences Europe GmbH, Eppelheim, Germany). The effluent cells were enriched by negative selection of T cells using nanobeads (Miltenyi Biotec) according to manufacturer’s instruction. The final purity of T cells (CD3⁺) was >95% as determined by flow cytometry.

Hojjat-Farsangi M., Jeddi-Tehrani M., Daneshmanesh A.H., Mozaffari F., Moshfegh A., Hansson L., Razavi S.M., Sharifian R.A., Rabbani H., Österborg A., Mellstedt H, & Shokri F. (2015). Spontaneous Immunity Against the Receptor Tyrosine Kinase ROR1 in Patients with Chronic Lymphocytic Leukemia. PLoS ONE, 10(11), e0142310.

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Isolation of Spleen and Peyer's Patch Lymphocytes

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Lymphocytes from spleen and Peyer's patches were isolated as previously described16 (link). Briefly, mouse spleen was homogenized and followed by treatment with ACK lysis buffer to lyses red blood cells. The single-cell suspension was collected by passing splenocytes through 70 μm cell strainers (BD Biosciences, Bedford, MA, USA). Then, the cell suspension was incubated in nylon-wool columns (Polysciences, Warrington, PA, USA) at 37 °C for 1 h and the enriched T cells were washed through the columns with complete RPMI-1640. To isolate lymphocytes from Peyer's patches, visible Peyer's patches were collected from ice-cold PBS-flushed small intestines. After homogenization, cells were passed through 70 μm cell strainers and lymphocytes were isolated by Ficoll gradient separation (GE Healthcare).

Zhong Z., Zhai Y., Bu P., Shah S, & Qiao L. (2017). Papilloma-pseudovirus eradicates intestinal tumours and triples the lifespan of ApcMin/+ mice. Nature Communications, 8, 15004.

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T-Cell Proliferation Assay with DCs

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WT and SHB^KD imDCs were matured by culturing with LPS (200 ng/mL) for 24 hours. Before harvest, WT or SHB^KD mature DCs (mDCs) were pulsed with 1 µg/mL OVA peptide (OVA_323-339) for 1 hour. The OVA-pulsed mDCs were washed three times with cold phosphate-buffered saline (PBS) and then used for T-cell proliferation assays. T cells were isolated from the spleen of OT-2 transgenic mice as described previously [6 (link)]. T cells purified on nylon wool columns (Poly Sciences, Warrington, PA, USA) were labeled with CFSE (1 µM). These CFSE-labeled T cells were co-cultured with OVA peptide–pulsed DCs at different ratios (1:5, 1:10, and 1:20) for 4 days. T cells were gated, and calculations were performed using the formula for proliferation index (PI): PI=1,000/geometric sum of gated CFSE. The T-cell proliferation capacity of OVA peptide–pulsed DCs was represented by the fold increase over the PI of the T cells co-cultured with unpulsed DCs.

Ahmed M.S., Kang M.H., Lee E., Park Y., Jeong Y, & Bae Y.S. (2017). SH2 domain–containing adaptor protein B expressed in dendritic cells is involved in T-cell homeostasis by regulating dendritic cell–mediated Th2 immunity. Clinical and Experimental Vaccine Research, 6(1), 50-60.

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