To prepare human PBMC-derived DCs, human monocytes were enriched by plastic adherence of PBMCs in a 100 mm Petri dish at 37 °C and 5% CO2. After 2 h of incubation, the nonadherent cells were reserved to sort CD8+ T cells with a Naive CD8+ T Cell Isolation Kit (Miltenyi Biotec, Cat: 130–093-244) for subsequent experiments. The remaining adherent cells were cultured in medium containing 100 ng/mL human GM-CSF (PeproTech, Cat: AF-300–03-50UG) and 20 ng/mL human IL-4 (InvivoGen, Cat: rcyec-hil4). After 48 h, the medium was refreshed, and 10 ng/mL TNF-α (InvivoGen, Cat: rcyc-htnfa) was added. On day 3, the human DC suspension was harvested.
Naive cd8 t cell isolation kit
Manufactured by Miltenyi Biotec
Sourced in United States
The Naive CD8+ T Cell Isolation Kit is a laboratory equipment product designed for the isolation of naive CD8+ T cells from various biological samples. It enables the efficient separation and enrichment of this specific cell population without the need for extensive sample preparation or manipulation.
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Lab products found in correlation
Naive cd4 t cell isolation kit, by Miltenyi Biotec (4 mentions)
Anti cd3 cd28 dynabeads, by Thermo Fisher Scientific (2 mentions)
Red blood cell lysis buffer, by Thermo Fisher Scientific (2 mentions)
Robo buffer, by STEMCELL (2 mentions)
Ls column, by Miltenyi Biotec (2 mentions)
Quadromacs separator, by Miltenyi Biotec (2 mentions)
Rpmi 1640 medium, by Corning (2 mentions)
Human gm csf, by Thermo Fisher Scientific (1 mentions)
Tnf α, by InvivoGen (1 mentions)
Siinfekl peptide, by Biosynth (1 mentions)
Automacs pro, by Miltenyi Biotec (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Facsaria 3, by BD (1 mentions)
Cd25 pe, by BD (1 mentions)
Sodium pyruvate, by Fujifilm (1 mentions)
Sodium pyruvate, by Nacalai Tesque (1 mentions)
2 mercaptoethanol, by Fujifilm (1 mentions)
Mem non essential amino acids, by Nacalai Tesque (1 mentions)
Anti tcr β mab, by BioLegend (1 mentions)
L glutamine, by Nacalai Tesque (1 mentions)
11 protocols using naive cd8 t cell isolation kit
1
Generation of Human PBMC-Derived Dendritic Cells
2
Adoptive Transfer of Activated OT-I Cells
3
Generation of NY-ESO-1-specific CD8+ T Cells
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A high-affinity NY-ESO-1 specific HLA-A*02-restricted TCR gene was transduced, 19305DP (19,305-TCR) [25 (link)]. CD8+ T cells were isolated from cryopreserved healthy-donor blood mononuclear cells (PBMC) using a Naive CD8 + T-cell Isolation-Kit (Miltenyi Biotec 130–093-244). The isolated cells (2.0 × 106 cells/well) < space removed were plated in complete RPMI supplemented with recombinant human IL-2* 300 IU/ml (Peprotech 200–02) and anti-CD3/CD28 dynabeads (1:3 to 1:1, bead:cell) (Thermo Fisher Scientific 11161D) in 6-well culture plates. After 48 h, T cells were harvested and seeded onto plates coated with recombinant retrovirus from PG13 supernatant. After transduction, T cells were de-adhered from dynabeads and re-suspended at (0.5–0.7 × 106/ml) in complete RPMI with IL-2. *Media supplemented with IL-2, IL-7, and IL-15 (10 ng/ml, Peprotech 200–02, 200–07, 200–15) was used for transduction of PBMC and naive CD8+ T-cells.
4
Adoptive Transfer of Naive OT-I and OT-II T Cells
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Naive CD62L+CD8+ T cells were purified from spleen cells of OT‐I mice using the naive CD8+ T Cell Isolation Kit and autoMACS Pro (Miltenyi Biotec). Naive CD62L+CD4+ T cells were purified from spleen cells of OT‐II mice using the naive CD4+ T Cell Isolation Kit and autoMACS Pro (Miltenyi Biotec). The purity of the CD62L+CD8+ and CD62L+CD4+ T cells was routinely assessed at >95%. At 1 day before immunization, a mixture of both cells (1 × 105) in 200 μl PBS was injected intravenously.
5
Isolation of Naive CD8+ T Cells
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De-identified whole-adult (18–55 years of age) and cord blood (39–41 weeks gestation) samples, each from three healthy donors, were obtained from New York Blood Center and National Disease Research Interchange, respectively. Mononuclear cells were isolated using Ficoll-paque Plus (GE Healthcare); CD8+ T cells were then isolated using a Naive CD8+ T Cell Isolation Kit (Miltenyi Biotec).
6
Naive CD8+ T cell Activation Assay
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Naive CD8+ T cells were purified from the spleen and peripheral lymph nodes of OT-I mice. Briefly, the spleen was perfused with RPMI 1640 supplemented with 10% FBS, red blood cells were lysed and then enriched by negative selection of naive CD8+ T cells using a commercial kit (Naive CD8+ T cell isolation kit- Miltenyi Biotec) following the manufacturer's instructions. Subsequently, naive CD8+ T cells (CD8+/CD44lo/ CD62Lhi/CD25−) were enriched (up to 99% purity) by cell-sorting isolation using Aria III FACS, BD Biosciences. The cells were then labeled with 5 μM of CellTrace Violet fluorescent probe (Invitrogen Corporation, Carlsbad, CA, USA) following the manufacturer's recommendations. Once labeled with the probe, 1 x 105 naive CD8 + OT-I T cells were incubated with 1 x 105 BALB/c BMDCs previously incubated for 72 h with E-ACs or P2X7 E-ACs loaded or not with SIINFEKL (OVA257-264). As controls, 1 x 105 T CD8 OT-I were incubated with 1 x 105 DCs C57BL/6 loaded with SIINFEKL peptide, or naive CD8+OT-I T lymphocytes were incubated with 2 nM of SIINFEKL peptide. The antigen-specific T cell response was measured by detection of CD25 in lymphocytes with the anti-mouse antibody CD25-PE (BD Biosciences, Clone: PC61.5) and by analysis of proliferation rounds as Cell Trace violet dilution using a cell sorter FACS Aria III and the software FlowJo 10.4.
7
Naive CD8 T Cell Activation and Glutamine Deprivation
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Naive CD8 T (CD44lowCD62Lhigh) cells were prepared using a Naive CD8+ T-cell Isolation kit (cat#130-096-543; Miltenyi Biotec, San Diego, CA, USA). Naive CD8 T cells (1.5 × 106) were stimulated with immobilized anti-TCR-β mAb (3 μg/ml, H57-597; BioLegend) and anti-CD28 mAb (1 μg/ml, 37.5; BioLegend) for 2 days in the presence of IL-2 (10 ng/ml, Pepro Tech). The cells were then transferred to a new plate and further cultured in the presence of IL-2 (10 ng/ml). The cells were cultured in RPMI 1640 with l -glutamine (cat#189-02025; Wako Chemicals) supplemented with 10% fetal bovine serum, 2 mM l -glutamine (16948-04; Nacalai Tesque, Kyoto, Japan), 1 mM sodium pyruvate (cat#06977-34; Nacalai Tesque), 1% MEM nonessential amino acids (cat#06344-56; Nacalai Tesque), 10 mM HEPES (cat#15630-080; Thermo Fisher Scientific, Waktham, MA, USA), 55 μM 2-Mercaptoethanol (cat#21985-023; Thermo Fisher Scientific), and 1% penicillin-streptomycin (cat#26253-84; Nacalai Tesque). For glutamine-deprived conditions, the cells were cultured in RPMI 1640 without l -glutamine (cat#183-02165; Wako Chemicals) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 1% MEM nonessential amino acids, 10 mM HEPES, 55 μM 2-Mercaptoethanol, and 1% penicillin-streptomycin.
8
Isolation and Labeling of Murine Dendritic Cells and T Cells
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Bone marrow cells were harvested from the tibia and femur of mice, and they were pre-enriched for DC differentiation by removing CD3 + , Gr-1 + and B220 + cells using biotinylated anti-Gr-1 (RB6-8C5), biotinylated anti-CD3 (17A2) and biotinylated anti-B220 (RA3-6B2) antibodies (BioLegend) with magnetic streptavidin MACS beads (Miltenyi). The pre-enriched bone marrow cells were cultured with 20 ng/mL GM-CSF (PeproTech) for 7–10 days, and harvested from the flasks and enriched for CD11c + bone marrow-derived dendritic cells (BMDCs) using CD11c MACS beads (Miltenyi) on the final day of culture. BMDCs were stained with a deep red cell tracker (Thermo Fisher). The spleen was harvested from mice (between 7 and 10 weeks of age), and T cells were purified from splenocytes using a naive CD8+ T cell isolation kit (Miltenyi), CD8+ T cell isolation kit (Miltenyi), or naive CD4+ T cell isolation kit (Miltenyi). T cells were stained with 2 µM calcein (Thermo Fisher).
9
Engineered NY-ESO-1 Specific TCR-T Cells
10
Isolation of Naive T Cells from Murine Tissues
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