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Na ve t cell isolation kit

Manufactured by Miltenyi Biotec
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The Naïve T cell isolation kit is a laboratory product designed to isolate naïve T cells from a mixed cell population. It utilizes an immunomagnetic separation technique to selectively remove non-naïve T cell subsets, allowing for the enrichment of the naïve T cell fraction.

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Lab products found in correlation

Taqman gene expression assay kit, by Thermo Fisher Scientific (2 mentions) Ficoll paque plus, by GE Healthcare (2 mentions) L929 cell, by American Type Culture Collection (1 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions) Macrophage isolation kit, by Miltenyi Biotec (1 mentions) Rhil 1β, by R&D Systems (1 mentions) Rhtgf β, by Thermo Fisher Scientific (1 mentions) Rhil 23, by R&D Systems (1 mentions) Rhil 6, by R&D Systems (1 mentions) Sodium pyruvate, by Thermo Fisher Scientific (1 mentions) Rhil 2, by Thermo Fisher Scientific (1 mentions) Soluble anti cd28, by Thermo Fisher Scientific (1 mentions) Ack buffer, by Thermo Fisher Scientific (1 mentions) Nebnext ultra rna library kit, by Illumina (1 mentions)

Spelling variants of this product

Isolation kits (10 citations) Cell isolation kits (5 citations) Mouse B cell and naïve T cell isolation kits (2 citations)

9 protocols using na ve t cell isolation kit

1

Isolation and Analysis of Immune Cells

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L929 cells were ordered from the ATCC (Manassas, USA). The Macrophage Isolation Kit and naïve T cell Isolation Kit was ordered from Miltenyi Biotec (Bergisch Gladbach, Germany). The plasmid and lentiviral were purchased from Hanheng Bio (Shanghai, China). Si-RNA was ordered from GenePharma (Shanghai, China). Lipofectamine 2000 reagent was purchased from Thermo Fisher (Haverhill, USA). The anti-mouse/human Roquin-1antibody was ordered from Invitrogen (California, USA). The primary mouse anti-mouse F4/80/CD11b/CX3CR1/Clec4F antibody and secondary antibody in immunofluorescence stains were purchased from Novus (Cambridge, USA). The antibodies in the flow cytometer were ordered from Biolegend (San Diego, USA). The antibodies and reagents in Western bolt analysis and immunoprecipitation were purchased from Abcam (Cambridge, USA). The primer and regents in qRT-PCR and Northern blot were ordered from Vazyme (Nanjing, China). ELISA kits were ordered from LiankeBio (Nanjing, China). The EVs and subcellular fraction isolation reagents were purchased from Beckman (Irvine, USA). The STAT3 inhibitor and AMPKα activator/inhibitor were ordered from MCE (Monmouth Junction, USA).
Zheng L., Ling W., Zhu D., Li Z., Li Y., Zhou H, & Kong L. (2023). Roquin-1 resolves sepsis-associated acute liver injury by regulating inflammatory profiles via miRNA cargo in extracellular vesicles. iScience, 26(8), 107295.
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2

Inducing Th9 Differentiation from Naive T Cells

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Human peripheral blood mononuclear cells were provided by healthy volunteers and obtained from the NIH Department of Transfusion Medicine (DTM) through their approved protocol number: NCT000001846. These blood samples were provided by the DTM on a de-identified basis. A signed informed consent was obtained from all donors. Naïve CD4+ T cells were purified by naïve T cell isolation kit (Miltenyi Biotec). Naïve T cells were cultured with plate-bound anti-human CD3 (1 μg/mL) and soluble anti-human CD28 (1 μg/mL) with the combination of TGF-β1 (2 ng/mL), IL-4 (10 ng/mL), anti-human IL-4 (10 μg/mL), TGF-βR inhibitor (SB431542, 5 μM), and TAK1 inhibitor (50 nM) for 48 h. mRNA expression were measured according to the protocol of TaqMan gene expression assay kits (Applied Biosystems) with following primers: Gapdh, Hs99999905_m1; Il9, Hs00914237_m1; Id3, Hs00954037_g1; Gata3, Hs00231122_m1. Id3 knockdown were performed by using Amaxa Human T cell Nucleofector kit with Id3-specific siRNAs (SI04431714) or AllStars Negative Control siRNA (Qiagen).
Nakatsukasa H., Zhang D., Maruyama T., Chen H., Cui K., Ishikawa M., Deng L., Zanvit P., Tu E., Jin W., Abbatiello B., Goldburg N., Chen Q., Sun L., Zhao K, & Chen W. (2015). The DNA-binding inhibitor Id3 regulates IL-9 production in CD4+ T cells. Nature immunology, 16(10), 1077-1084.
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3

Differentiation of Naïve CD4+ T Cells into Th17 Cells

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Human peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coats of participants by Ficoll-paque Plus (GE Healthcare catalog #17-1440-02). Naïve CD4+ T cells were isolated from the PBMCs using a naïve T cell isolation kit (Miltenyi Biotec catalog # 130-094-131) per manufacturer's instruction. CD4+ naïve T cells were activated using an activation/expansion kit (Miltenyi Biotec catalog #130-091-441) with anti-CD3 and anti-CD28 bound to bead particles at the ratio of 1 bead particle per 2 cells. CD4+ T cells were cultured differentiated to become Th17 cells with hIL-2 (10ng/ml), rhIL-1β (10ng/ml), rhTGF-β (1ng/ml), rhIL-6 (10ng/ml), rhIL-23 (10ng/ml), anti-IFN-γ (10μg/ml), and/or anti-IL-4 (10μg/ml) in T cell culture media. T cell culture media was RPMI containing 10% FBS, 1% penicillin/streptomycin, 2mM l-glutamine, 10mM HEPES, 0.1mM non-essential amino acids, and 1mM sodium pyruvate (Gibco, Carlsbad, CA). All antibodies and rhIL-1β, rhIL-6, and rhIL-23 were purchased from R&D Systems. rhIL-2 and rhTGF-β was purchased from PeproTech (Rocky Hill, NJ).
Newcomb D.C., Cephus J.Y., Boswell M.G., Fahrenholz J.M., Langley E.W., Feldman A.S., Zhou W., Dulek D.E., Goleniewska K., Woodward K.B., Sevin C.M., Hamilton R.G., Kolls J, & Peebles RS J.r. (2015). Estrogen and progesterone decrease Let-7f miRNA expression and increase IL-23/IL-23R signaling and IL-17A production in severe asthma. The Journal of allergy and clinical immunology, 136(4), 1025-1034.e11.
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4

Inducing Th9 Differentiation from Naive T Cells

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Human peripheral blood mononuclear cells were provided by healthy volunteers and obtained from the NIH Department of Transfusion Medicine (DTM) through their approved protocol number: NCT000001846. These blood samples were provided by the DTM on a de-identified basis. A signed informed consent was obtained from all donors. Naïve CD4+ T cells were purified by naïve T cell isolation kit (Miltenyi Biotec). Naïve T cells were cultured with plate-bound anti-human CD3 (1 μg/mL) and soluble anti-human CD28 (1 μg/mL) with the combination of TGF-β1 (2 ng/mL), IL-4 (10 ng/mL), anti-human IL-4 (10 μg/mL), TGF-βR inhibitor (SB431542, 5 μM), and TAK1 inhibitor (50 nM) for 48 h. mRNA expression were measured according to the protocol of TaqMan gene expression assay kits (Applied Biosystems) with following primers: Gapdh, Hs99999905_m1; Il9, Hs00914237_m1; Id3, Hs00954037_g1; Gata3, Hs00231122_m1. Id3 knockdown were performed by using Amaxa Human T cell Nucleofector kit with Id3-specific siRNAs (SI04431714) or AllStars Negative Control siRNA (Qiagen).
Nakatsukasa H., Zhang D., Maruyama T., Chen H., Cui K., Ishikawa M., Deng L., Zanvit P., Tu E., Jin W., Abbatiello B., Goldburg N., Chen Q., Sun L., Zhao K, & Chen W. (2015). The DNA-binding inhibitor Id3 regulates IL-9 production in CD4+ T cells. Nature immunology, 16(10), 1077-1084.
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5

Atorvastatin Modulates Dendritic Cell-Induced T Cell Proliferation

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Immature dendritic cells derived from bone marrow of beta-actin RFP-transgenic C57BL/6 mice generated in the absence or presence of atorvastatin were incubated for 3 h with OVA323-339 peptide (100 µg/ml) and then injected intracutaneously into Rag1-ko mice. Naïve CD4 T cells were isolated from the spleen and lymph node cells of OT-II animals by magnetic cell sorting, using the Naïve T cell isolation kit (Miltenyi Biotec), and transferred to the Rag1-ko mice one day after the iDC. After 5 days cells were isolated from the draining lymph nodes, restimulated in vitro with varying concentrations of OVA-peptide or control stimuli (anti-CD3/CD28, concavalin A), and proliferation was measured in a standard 3H-thymidine incorporation assay.
Leuenberger T., Pfueller C.F., Luessi F., Bendix I., Paterka M., Prozorovski T., Treue D., Luenstedt S., Herz J., Siffrin V., Infante-Duarte C., Zipp F, & Waiczies S. (2014). Modulation of Dendritic Cell Immunobiology via Inhibition of 3-Hydroxy-3-Methylglutaryl-CoA (HMG-CoA) Reductase. PLoS ONE, 9(7), e100871.
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6

Proliferation of OVA-specific T cells

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Immature dendritic cells (iDC and aiDC) were incubated with OVA323–339-peptide for 30 min at 37°C. Cells were then harvested, washed thoroughly, and cocultured with CFSE-labeled OVA-specific naïve T cells isolated from OT-II transgenic mice by magnetic cell sorting, using the Naïve T cell isolation kit (Miltenyi Biotec). The ratio of iDC/aiDC to T cells was 1:10. After 72 hours cells were harvested, T cell markers were stained (CD4-AF647, vα2-Bio, streptavidin-PacificBlue) and T cell proliferation was measured by FACS as a decrease in CFSE-intensity.
Leuenberger T., Pfueller C.F., Luessi F., Bendix I., Paterka M., Prozorovski T., Treue D., Luenstedt S., Herz J., Siffrin V., Infante-Duarte C., Zipp F, & Waiczies S. (2014). Modulation of Dendritic Cell Immunobiology via Inhibition of 3-Hydroxy-3-Methylglutaryl-CoA (HMG-CoA) Reductase. PLoS ONE, 9(7), e100871.
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7

Isolation and Differentiation of Naïve CD4+ T Cells

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Whole blood leukopaks from healthy adult donors were purchased from the Pittsburgh Central Blood Bank, with the approval of the University of Pittsburgh Institutional Review Board. PBMCs were isolated from the buffy-coat by Ficoll-Paque Plus (GE Healthcare Bio-Science AB) density gradient centrifugation. CD4+ T cells were isolated with the naïve T Cell Isolation Kit (Miltenyi Biotec Inc., Bergisch Gladbach, Germany). The isolated naïve CD4+ T cells were cultured in 96-well plates at a density of 4 × 104 cells/well. For Th0 cell cultures, cells were activated with 10 μg/mL plate-bound human anti-CD3 (PeproTech, Cranbury, NJ) and 1 μg/mL soluble anti-CD28 (PeproTech, catalog 10311–20) for 5 days under nonpolarizing conditions. For Th1 or Th17 cell differentiation, cells were cultured utilizing Human Th1 or Th17 Cell Differentiation Kit (R&D Systems, Inc.) according to the manufacturer's instructions.
Wang P., Killeen M.E., Sumpter T.L., Ferris L.K., Falo LD J.r., Freeman B.A., Schopfer F.J, & Mathers A.R. (2021). Electrophilic nitro-fatty acids suppress psoriasiform dermatitis: STAT3 inhibition as a contributory mechanism. Redox Biology, 43, 101987.
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8

Isolation and Activation of Naive CD4+ T Cells

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Naïve T cells were isolated from the spleens of 8-12 week old C57 males using the Miltenyi Biotec Naïve T cell isolation kit. Spleens were crushed between frosted glass slides and filtered through 70 μm cell strainers. Red blood cells were lysed using ACK buffer (Thermo Fisher Scientific). The cell pellet was incubated first with a biotinylated antibody cocktail containing antibodies against CD8a, CD11b, CD11c, CD19, CD25, CD45R (B220), CD49b, CD115, MHCII, TER119, and TCRγ/δ, then anti-biotin magnetic beads. The cells were then passed through a MACS LS column and untouched CD4+ naïve T cells were collected and resuspended in RPMI 1640 supplemented with 10% FBS and 1× Pen-Strep. Naive Cells were incubated for 1 hour at 37°C before being transferred to wells surface coated with 3 μg/mL anti-mouse CD3ε. 5*105 CD4+ T cells were added to each well, supplemented with 2 μg/mL anti-mouse CD28. Blank or IL-33 scaffolds were added to wells and cells were incubated 72 hours at 37°C. Cell culture supernatants were spun down to remove cells, snap-frozen on liquid nitrogen, and stored at −80°C until analysis. Samples were sent to the University of Michigan ELISA core for analysis of IL-13.
Liu J.M., Zhang X., Joe S., Luo X, & Shea L.D. (2018). Evaluation of biomaterial scaffold delivery of IL-33 as a localized immunomodulatory agent to support cell transplantation in adipose tissue. Journal of immunology and regenerative medicine, 1, 1-12.
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9

Transcriptomic Profiling of Naive CD4+ T Cells

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Total naïve CD4+ T cells were isolated from the spleen of WT and Pcbp1f/f Cd4-Cre mice using the naïve T cell isolation kit (Miltenyi, USA). Total RNA was extracted from the cells and sent to Novogene for RNA-seq analysis. Libraries were prepared using 50 ng of total RNA using the NEBNext Ultra RNA Library Kit for Illumina (catalog no. E7530) and sequenced on HiSeq 3000 at 75 base pair paired-end. Each sample was analyzed in quadruplet. Sequencing reads were first evaluated for quality control using the FastQC software and then aligned against the mouse mm10 reference genome using the TopHat2 software. Mapped reads were assigned to gene features and quantified using the HTSeq software and further evaluated for quality control using correlation and multidimensional scaling plots. Normalization and differential expression were performed using the edgeR software. The significantly DEGs (FDR < 0.05) were visualized using heat maps and volcano plot representations and considered for subsequent functional enrichment analysis using ToppGene Suite.
Ansa-Addo E.A., Huang H.C., Riesenberg B., Iamsawat S., Borucki D., Nelson M.H., Nam J.H., Chung D., Paulos C.M., Liu B., Yu X.Z., Philpott C., Howe P.H, & Li Z. (2020). RNA binding protein PCBP1 is an intracellular immune checkpoint for shaping T cell responses in cancer immunity. Science Advances, 6(22), eaaz3865.
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