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Naive t cell isolation kit

Manufactured by Miltenyi Biotec
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Sourced in Germany

The Naive T cell isolation kit is a laboratory tool used for the separation and purification of naive T cells from a mixed cell population. The kit utilizes a magnetic bead-based approach to selectively isolate the desired cell type. The core function of this product is to provide a reliable and efficient method for the isolation of naive T cells for downstream applications and research purposes.

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Lab products found in correlation

Anti cd3 cd28 beads, by Thermo Fisher Scientific (1 mentions) Polybrene, by Santa Cruz Biotechnology (1 mentions) Cd3 cd28 dynabeads, by Miltenyi Biotec (1 mentions) Recombinant il 2, by Miltenyi Biotec (1 mentions) Cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions) Rbc lysis buffer, by Abcam (1 mentions) Mitomycin c, by Merck Group (1 mentions) Recombinant il 3, by BioLegend (1 mentions)

4 protocols using naive t cell isolation kit

1

Dendritic Cell Vaccine Generation

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PBMCs were routinely isolated, and DCs were induced from PBMCs and cultured. DCs were infected with rAAV/AFP 1 day after culture (DC-rAAV/AFP), and DC precursors were sensitized with 100 µg Dex (DC-Dex) 5 days after culture to prepare DC vaccines. DC-rAAV/AFP, DC-Dex, and non-transfected DCs after 7 days of induction were adjusted to a density of 1×105 cells/mL and incubated with 25 µg/mL mitomycin C at 37°C for 45 minutes. After being washed three times in PBS, cells were resuspended in RPMI 1640 medium. DC-rAAV/AFP (Group A), Dex (Group B), DC-Dex (Group C), and non-transfected DCs (Group D) were mixed with naive T cells, which were isolated by negative selection using Naive T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer’s instructions, at a ratio of 1:10, respectively. Cells in Group B (containing 1×106 naive T cells per well) were co-incubated with 100 µg/well Dex at 37°C with 5% CO2 for 10 days.
Li J., Huang S., Zhou Z., Lin W., Chen S., Chen M, & Ye Y. (2018). Exosomes derived from rAAV/AFP-transfected dendritic cells elicit specific T cell-mediated immune responses against hepatocellular carcinoma. Cancer Management and Research, 10, 4945-4957.
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2

Adoptive Transfer of Regulatory T Cells

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Primary CD4+CD25+ nTreg cells from WT and Nfat2fl/fl x Cd4cre mice were activated with anti-CD3/CD28 beads (Invitrogen) for 3 d in presence of 50 U/ml IL-2. Beads were removed and 5 × 106 nTreg cells were collected and resuspended in 1 ml fresh RPMI medium containing 8 µg/ml polybrene (Santa Cruz Biotechnology) and 50 U/ml lL-2 (PeproTech). After 30 min, cells were centrifuged and the supernatant was mostly removed. Cell pellets were resuspended in 0.25 ml/2.5 × 106 IFU ready-made lentiviral solution (gfp or Cxcr5-gfp [pLenti-EF1a(mCXCR5)-Rsv(GFP-Puro)]; Amsbio) and transduction was performed by spin infection (1,800 rpm for 90 min at 32°C). Then cells were diluted in 2 ml RPMI medium containing 50 U/ml lL-2 (PeproTech) and incubated for additional 4 h at 37°C. In the meantime, CD4+CD25–CD62Lhi T cells and B cells were isolated from WT mice using the naive T cell isolation kit and the untouched B cell isolation kit, respectively (Miltenyi Biotec). nTreg cells were washed twice and 4 × 105 transduced WT or NFAT-deficient nTregcells along with 5 × 106 B cells and 2 × 106 naive T cells were adoptively transferred i.p. into Rag1−/− mice. After 14 d, mice were immunized with 100 µg NP-KLH and analyzed on day 21.
Vaeth M., Müller G., Stauss D., Dietz L., Klein-Hessling S., Serfling E., Lipp M., Berberich I, & Berberich-Siebelt F. (2014). Follicular regulatory T cells control humoral autoimmunity via NFAT2-regulated CXCR5 expression. The Journal of Experimental Medicine, 211(3), 545-561.
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3

In Vitro T-Reg Suppression Assay

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For in vitro Treg suppression assays63 (link), red blood cell lysis was performed using RBC lysis buffer (Abcam) on splenocytes of Foxp3Cre-YFP mice at room temperature for 2 min. CD4+ T cells were enriched using a CD4+ T cell isolation kit (Miltenyi), and Treg cells were then purified by FACS. Treg cells were cultured and preactivated in vitro in the presence of 2,000 U ml–1 recombinant IL-2 (Miltenyi) and CD3/CD28 Dynabeads for 3 d (ref. 64 ) to induce eTreg cell differentiation.
Antigen-presenting cells were enriched by depleting CD90.2+ splenocytes, exposed to 20 µg ml–1 mitomycin C (Sigma) for 30 min at 37 °C and washed five times with PBS. Antigen-presenting cells were plated at 2 × 105 cells per well and used for co-stimulation, and 1 µg ml–1 anti-CD3 (clone 17A2) was added for polyclonal TCR activation. CD4+ T cells were isolated using a naive T cell isolation kit (Miltenyi) and labeled with CellTrace Violet according to the manufacturer’s protocol, and 2.5 × 104 cells were seeded per well in 96-well plates. Treg cells were added at the indicated ratios, and the assay was performed for 72 h. For antibody treatment, 10 µg ml–1 anti-mouse IL-23R (clone 12B2B64) was added. Percent suppression was assessed based on the division index (DI) calculated in FlowJo with the following formula: percent suppression = 100 – (DITreg:Tcon ratio/DITcon alone) × 100 (ref. 65 (link)).
Wertheimer T., Zwicky P., Rindlisbacher L., Sparano C., Vermeer M., de Melo B.M., Haftmann C., Rückert T., Sethi A., Schärli S., Huber A., Ingelfinger F., Xu C., Kim D., Häne P., Fonseca da Silva A., Muschaweckh A., Nunez N., Krishnarajah S., Köhler N., Zeiser R., Oukka M., Korn T., Tugues S, & Becher B. (2024). IL-23 stabilizes an effector Treg cell program in the tumor microenvironment. Nature Immunology, 25(3), 512-524.
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4

Microglia and T cell activation assay

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Sorted microglia were cultured in complete medium (RPMI-1640 supplemented with 10%FBS, 2mM L-glutamine, 10Uml−1 penicillin and streptomycin, 10mM HEPES, 50μM 2-mercaptoethanol, 1mM sodium pyruvate and 1x nonessential amino acids) and kept in humidified 5% CO2 incubator at 37°C. Microglia were exposed to 20ng/ml recombinant IL-3 (Biolegend) and/or 2μg/ml Beta-Amyloid (1-42) HiLyte™ conjugated to pHrodo iFL red (Invitrogen) for 3 hours. T-cells. Naive T cells were isolated from the spleen and lymph nodes using a Naive T cell isolation kit (Miltenyi Biotec) and cultured on anti-CD3 (2 μg/mL) coated plates in the presence of soluble anti-CD28 (2 μg/mL) and rmIL-2 (10 μg/mL) for 3 days and re-stimulated with PMA (100 ng/mL) and ionomycin (500 ng/mL) in the presence of GolgiPlug and GolgiStop (1:1000) for 3.5 hours prior to cell surface staining and analysis.
McAlpine C.S., Park J., Griciuc A., Kim E., Choi S.H., Iwamoto Y., Kiss M.G., Christie K.A., Vinegoni C., Poller W.C., Mindur J.E., Chan C.T., He S., Janssen H., Wong L.P., Downey J., Singh S., Anzai A., Kahles F., Jorfi M., Feruglio P.F., Sadreyev R.I., Weissleder R., Kleinstiver B.P., Nahrendorf M., Tanzi R.E, & Swirski F.K. (2021). Astrocyte-derived interleukin-3 reprograms microglia and limits Alzheimer’s disease. Nature, 595(7869), 701-706.
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