For purified human naïve CD4+ T cells, a human naïve CD4+ T cell isolation kit was used according to the manufacturer’s instructions (Miltenyi Biotec). Human naïve CD4+ T cells (1 × 105 cells) and SH-10-TC cells (3 × 104 cells) were cultured at 37°C in RPMI 1640 (Lonza) containing 10% fetal bovine serum (Heat inactivated; GeminiBio), penicillin/streptomycin, and 50 μM 2-mercaptoethanol (Sigma Aldrich) in the presence of CD3/28 Dynabeads (Thermo-Fisher Scientific) for 48 h in the presence or absence of 5 μM GO-Y022 or 5 mM 2DG.
Human na ve cd4 t cell isolation kit
Manufactured by Miltenyi Biotec
Sourced in Germany
The Human Naïve CD4+ T Cell Isolation Kit is a lab equipment product designed for the isolation of naïve CD4+ T cells from human peripheral blood mononuclear cells (PBMCs). The kit utilizes a magnetic separation-based method to negatively select the target cell population.
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Lab products found in correlation
Cd3 28 dynabeads, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Rpmi 1640, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Gemini Bio (1 mentions)
Human cd4 cd25 treg isolation kit, by Miltenyi Biotec (1 mentions)
Fluorescence activated cell sorter, by BD (1 mentions)
Anti cd28, by Thermo Fisher Scientific (1 mentions)
T cell expansion medium, by Thermo Fisher Scientific (1 mentions)
Mirna 374b 5p inhibitor, by Qiagen (1 mentions)
Mirna 374b 5p mimic, by Qiagen (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Hiperfect transfection reagent, by Qiagen (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Human cd4 microbeads, by Miltenyi Biotec (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Beyotime (1 mentions)
Facsaria, by BD (1 mentions)
Human pan dc enrichment kit, by Miltenyi Biotec (1 mentions)
9 protocols using human na ve cd4 t cell isolation kit
1
Naïve CD4+ T Cell Activation Assay
2
Isolation of Human Naïve CD4+ T Cells
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Human naïve CD4+ T cells were isolated as previously described [21 (link)], using human naïve CD4+ T-cell isolation kit (Miltenyi Biotech).
3
Isolation and Purification of Naive CD4+ T Cells, Tregs and CD4+CD25- T Cells
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PBMCs were isolated from healthy fertile women as described previously.35 (link) Naïve CD4+ T cells, CD4+CD25+ Tregs and CD4+CD25− T cells were purified by MACS using the human naïve CD4+ T Cell Isolation Kit and the human CD4+CD25+ Treg Isolation Kit, respectively, according to the manufacturer's instructions (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The purity of the separated naïve CD4+ T cells, CD4+CD25+ T cells and CD4+CD25− T cells was >95%, as determined by flow cytometry.
4
Modulation of Th-17 Cell Differentiation
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EXAMPLE 11
Ex Vivo Reduction of Human Th-17 Cells Differentiation
Material and Methods
Peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteers using density gradient centrifugation and CD4+ naïve T cells were enriched from PBMCs using human naïve CD4+ T cell isolation kit (Miltenyi Biotech). Isolated CD4+ T cells were maintained in RPMI medium and differentiated toward Th-17 cells by stimulation with IL-12 and IL-2, both 5 ng/ml in combination with 1.5 ng/ml of antibodies anti CD3 and anti CD28 and 0.03 μM PGE2±Compound 1 at concentration of 0.01-0.03-0.1-0.3 μM for 48 hours.
At the end of incubation time cells were stained with fluorescence-conjugated antibodies specific for CD4, CCR6, CD45, CD25 and IL-17 (all from BD Bioscience). Finally, the number of Th-17 cells was determined by flow cytometry and the events were measured by Fluorescence Activated Cell Sorter (FACS; BD Bioscience) and analyzed with dedicated software.
Results
The results obtained are reported in
Conclusion
Data obtained provide evidence that Compound 1 negatively modulates Th-17 cells differentiation.
5
Isolation and Transfection of Naïve CD4+ T Cells
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Serum samples and peripheral blood mononuclear cells (PBMCs) were isolated from the EDTA-anticoagulated whole blood specimens of IBD patients and control subjects via Ficoll-Paque™ Plus density centrifugation (GE Healthcare Bio-Science, USA). Human-lymphocyte-cell-separation-medium kit (Solarbio Life Sciences, China) was used to further isolate PBMCs by following the kit’s protocol. Then, CD4+T cell isolation was performed by magnetically activated cell sorting using the human naïve CD4+T cell isolation kit (Miltenyi Biotec, Germany). Flow cytomerty was conducted to assess the purity of CD4+ T cells (> 95%). The cells were then cultured in T-cell expansion medium (Thermo-Fisher) containing 15% FBS and 100 g/mL penicillin–streptomycin. The naïve CD4+ T cells were assigned to miRNA-374b-5p mimic (50 nmol/mL; Qiagen, 219,600) group, miRNA-374b-5p inhibitor (200 nmol/mL; Qiagen, 219,300) group, negative control (NC) group and blank group, and transfected for 4 h with Hiperfect Transfection Reagent (Qiagen) by following the kit’s protocols. Finally, the transfected cells were exposed to 1 μg/mL anti-CD28 and 1 μg/mL anti-CD3(eBioscience) at 37 °C for 48 h.
6
Isolation and Culture of Naïve CD4+ T Cells
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Total CD4+ T cells were isolated from PBMCs using human CD4 microbeads (Miltenyi Biotec, Germany), and naïve CD4+ T cells were selected by human Naïve CD4+ T Cell Isolation Kit (Miltenyi Biotec). Purified cells were then cultured in RPMI 1640 medium (GIBCO, USA) supplemented with 10% fetal bovine serum (GIBCO), and 1% penicillin/streptomycin (Beyotime, China) at 37 °C with 5% CO2.
7
PBMC Isolation and DC-T Cell Co-culture
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PBMCs were isolated from 100 mL of heparinized peripheral venous blood using Ficoll density gradient centrifugation. Total PBMCs were equally divided for DC and for autologous T cell isolation. The total myeloid DC population was isolated and negatively selected using a human Pan-DC enrichment kit (Miltenyi Biotec B. V & Co. KG, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. Subsequently, the negatively selected DCs were stained with fluorochrome-conjugated antibodies for pDCs (Lin1−, CD11c−, CD123+) and mDCs (Lin1−, CD11c+, CD123−) (APC-CD11c-mAb, PE-CD123mAb, FITC-LIN1mAb-BD Biosciences) followed by FACS sorting (FACS ARIA- BD Bioscience, USA). Naïve CD4+ T cells were isolated via negative selection using a human naïve CD4+ T cell isolation kit (Miltenyi Biotec B. V & Co. KG, Germany) according to the manufacturer’s protocol and stored at 4 °C during the 24 h DC pretreatment until co-culture initiation.
8
Isolation of Naïve T-Cells from PBMC
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Peripheral blood mononuclear cells (PBMC) from healthy subjects were isolated from de-identified leukocyte reduction system (LRS) cones containing leukocyte rich whole blood from platelet donors at the University of Iowa, DeGowin Blood Center. PBMC isolation was performed with Ficoll-Paque (GE Healthcare) density gradient centrifugation and frozen in DMSO-containing media for further use. Naïve CD8+ T-cells and/or naïve CD4+ T-cells were isolated from freshly thawed PBMC [RPMI 1640 (Corning, 10-040-CV) with DNase at 10KU/ml (Sigma D4513-1vl)] with manual LS column MACS sorting using human naïve CD8+ T-cell isolation kit (Miltenyi Biotech 130-093-244) and human naïve CD4+ T-cell isolation kit (Miltenyi Biotech 130-094-131) respectively according to manufacturer specifications. Sort purities were routinely above 95% by flow cytometric analysis (Supplementary Figure 1A
9
Naïve CD4+ T Cell Differentiation into Th17 Cells
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Fresh whole blood was purchased from the La Jolla Institute for Allergy and Immunology (LJI) and used fresh on the day of the draw. Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll density gradient (Lymphopure). Isolated PBMCs were then sorted using a Human Naïve CD4 T cell Isolation kit (Miltenyi Biotech) according to the manufacturer's instructions. Isolated naïve CD4 T cells were plated in 96‐well plates at 100 000 cells per well in RPMI 1640 supplemented with NEAA, sodium pyruvate, and beta‐mercaptoethanol. To induce Th17 differentiation, IL‐6 (50 ng mL−1), TGF‐β1 (5 ng mL−1), IL‐23 (5 ng mL−1) and IL‐1β (20 ng mL−1), and IL‐21 (200‐21, 1019226) (25 ng mL−1) were added at the time of plating, with various concentrations of ATRA. All cytokines were recombinant human cytokines purchased from Peprotech and were the same as those used in mouse T cell differentiation assays, with the exception of IL‐21.
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