PBMCs were isolated within 12 h of sample collection and monocytes were removed by overnight plating in a culture dish. The next day, the CD4+CD45RA+ cells were purified using naïve CD4+ T-cell isolation kit II (product no. 130-094-131, Miltenyi Biotec). CD4+ T-cells were isolated using positive selection magnetic beads using isolation kit (product no. 130-045-101, Miltenyi Biotec). Purified naïve CD4+ T-cells represented a >97% pure population. Purified cells were maintained in culture with 20 U of IL-2 for two days. Thereafter, these cells were stimulated with plate-bound ICs at 10 μg/ml and purified soluble C5b-9 at 2.5 μg/ml for each 1X106 cells in the presence of plate-bound anti-CD3 at 0.25 μg/ml. Positive control cells were stimulated with plate-bound 2 μg/ml of anti-CD28 (clone 28.2) and 0.25μg/ml of anti-CD3 (eBioscience, clone OKT3). For inhibition, cells were cultured in 25nM of P505, a Syk inhibitor (product no. PRT06207, Sellkchem) and 50 μM of HCQ (product no. 263010250, Acros organics). Cells were cultured for 48 h in the presence of IL-2 (20 IU), for each one ml of medium (Peprotech). Post 48 h, cells were re-stimulated and processed for staining at 96 h.
Na ve cd4 t cell isolation kit 2
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
The Naïve CD4+ T Cell Isolation Kit II is a tool for the isolation of untouched naïve CD4+ T cells from human peripheral blood mononuclear cells (PBMCs). It uses a magnetic bead-based negative selection approach to enrich for the target cell population.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd28, by BD (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (4 mentions)
Dneasy blood and tissue kit, by Qiagen (3 mentions)
Cd14 microbead, by Miltenyi Biotec (3 mentions)
Ficoll paque plus, by GE Healthcare (3 mentions)
Anti cd3, by BD (3 mentions)
Tgf β, by R&D Systems (3 mentions)
Ez dna methylation kit, by Zymo Research (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (2 mentions)
Anti il 4, by Thermo Fisher Scientific (2 mentions)
Cd8 t cell isolation kit, by Miltenyi Biotec (2 mentions)
Celltrace cfse, by Thermo Fisher Scientific (2 mentions)
Percoll density gradient centrifugation, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Rosettesep, by STEMCELL (2 mentions)
Interleukin 6 (il 6), by R&D Systems (2 mentions)
Il 23, by R&D Systems (2 mentions)
Efluor 506, by Thermo Fisher Scientific (2 mentions)
46 protocols using na ve cd4 t cell isolation kit 2
1
Naïve CD4+ T-cell Stimulation Assay
2
Isolation of Naive CD4+ T Cells
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Ethical approval for this study was granted by the ethics committee of Kyoto University Graduate School and Faculty of Medicine. Written informed consent was obtained from all study participants. Tonsils were obtained from five patients (consisting of one female and four male patients ranging from 5 to 48 years old) having a tonsillectomy because of tonsillar hypertrophy. Tonsils were minced and digested with 2.5 mg/mL collagenase D (Roche) at 37°C for 1.5 h and then subsequently analyzed. These studies were exploratory research studies.
Fresh PBMCs from healthy volunteers were collected using Lymphocyte Separation Solution 1.077 (Nacalai Tesque) followed by cell separation and cell culture without cryopreservation. The age of healthy volunteers (consisting of five females and ten males) was ranging from 34 to 54 years old. Cell number was determined with counting chamber (TGK). The average yield of PBMC was 30 million cells per 10 mL peripheral blood with a viability of more than 95%. Naive blood CD4+ T cells were purified with naïve CD4+ T Cell isolation kit II (Miltenyi Biotec) through column twice as a fraction negative for CD8, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD45RO, CD56, CD123, TCRγ/δ, HLA‐DR, or CD235a and the purity of CD3+CD4+CD45RA+ cells in the sorted cells was more than 98%.
Fresh PBMCs from healthy volunteers were collected using Lymphocyte Separation Solution 1.077 (Nacalai Tesque) followed by cell separation and cell culture without cryopreservation. The age of healthy volunteers (consisting of five females and ten males) was ranging from 34 to 54 years old. Cell number was determined with counting chamber (TGK). The average yield of PBMC was 30 million cells per 10 mL peripheral blood with a viability of more than 95%. Naive blood CD4+ T cells were purified with naïve CD4+ T Cell isolation kit II (Miltenyi Biotec) through column twice as a fraction negative for CD8, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD45RO, CD56, CD123, TCRγ/δ, HLA‐DR, or CD235a and the purity of CD3+CD4+CD45RA+ cells in the sorted cells was more than 98%.
3
Naïve CD4+ T Cell Isolation
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From each study participant, 80 mL of whole blood was collected then subjected to Ficoll-gradient centrifugation (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) to isolate peripheral blood mononuclear cells. Naïve CD4+ T cells were then isolated from peripheral blood mononuclear cells by negative selection magnetic bead cell separation (indirect labelling) using the Naïve CD4+ T Cell Isolation kit II (Miltenyi Biotec, Cambridge, Massachusetts, USA). The purity of the isolated naïve CD4+ T cells was confirmed >95% using fluorochrome-conjugated antibodies targeting CD4 and CD45RA. DNA was then extracted using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, California, USA) and bisulfite converted using the EZ DNA Methylation Kit (Zymo Research, Irvine, California, USA) for subsequent DNA methylation analysis.
4
Generation of Human Monocyte-Derived Dendritic Cells
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Human mo-DCs were prepared as described previously [90 (link)]. Briefly, peripheral blood mononuclear cells (PBMCs) were obtained from healthy donor blood by Ficoll-Hypaque (GE Healthcare) density centrifugation. CD14+ cells (purity > 95%) were isolated using the EasySep™ Human CD14 Positive Selection Kit (STEMCELL) and cultured in RPMI-1640 medium supplemented with penicillin/streptomycin, 10% FBS, recombinant hGM-CSF (100 ng/ml) and hIL-4 (100 ng/ml; both from R&D) for 6 days. Human mo-DCs were treated with 10 µM SB203580 and HDM for 24 h, washed extensively and cocultured with human blood naïve CD4+ T cells isolated using the Naïve CD4+ T Cell Isolation Kit II (Miltenyi Biotec) at a ratio of 1:10. After 7 days of coculture, live T cells were purified and stimulated with plate-bound anti-human CD3 (UCHT1; BioLegend) for 5 h and then harvested for mRNA analysis. PBMCs were collected from allergic rhinitis (AR) patients and stimulated with the p38 inhibitor SB203580 or DMSO (vehicle) for 8 h, with GolgiStop added to the culture medium for the last 5 h. Cells were harvested, and IL-12p40 (C8.6, eBioscience) expression in DCs was detected by ICS. This study was approved by the Ethics Committee of the Eye & ENT Hospital of Fudan University (2017-0301). Informed consent was obtained from all volunteers.
5
Enrichment and Coculture of Naive CD4+ T Cells
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Through depletion of contaminating cells, untouched CD4+T cells (mean of 97% CD4+, detected by flow cytometry) were enriched from density gradient-enriched fraction of PBMC of the buffy coats using naïve CD4+T cell isolation kit II (Miltenyi Biotec). Isolated CD4+T cells were plated at 105 cells/well and cocultured with 104 prestimulated allogeneic MoLCs. After 5 days of coculture supernatants were harvested, cells were analyzed by flow cytometry.
6
Isolation of Human Mo-DCs and Naive CD4+ T Cells
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Human Mo-DCs and CD45RA+ naïve CD4+ T cells (>99% purity) were prepared as previously described [9 ]. Briefly, human Mo-DCs were prepared from human peripheral blood specimens (PBMCs) using positive selection with CD14 MicroBeads (Miltenyi Biotec). The Mo-DCs were then cultured in the presence of GM-CSF (50 ng/mL) and IL-4 (50 ng/mL) for five days. Human CD45RA+ naïve CD4+ T cells were prepared via negative selection using a naïve CD4+T-cell isolation kit II (Miltenyi Biotec, Auburn, U.S.A.). The study using PBMCs obtained from healthy volunteers was approved by the Saitama Medical University Ethics Committee.
7
Isolation of Naïve CD4+ T Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from 300 mL of whole blood from healthy donors after centrifugation on a Ficoll-Hypaque density gradient (GE Healthcare, Uppsala, SE). CD4+CD45RA+ naïve T cells (Tn) were isolated by negative selection, utilizing a naïve CD4+ T Cell Isolation Kit II with a QuadroMACS separator unit (MiltenyiBiotec, Bergisch Gladbach, DE) according to the manufacturer’s instructions. The purity of the isolated CD4+CD45RA+ cells was evaluated with a BD FACSCantoTM II flow cytometer and FACSDiva software (BD Biosciences, San Jose, CA, USA). Purity was above 97%, and CD4+CD25+FOXP3+ cells were below 2% for all donors.
8
Isolation and Culture of Naive CD4+ T Cells
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PBMCs were collected from whole (peripheral or cord) blood of consenting donors and cryopreserved. Upon thaw, naive CD4+ T cells were isolated by negative selection (Naïve CD4 T Cell Isolation Kit II, Miltenyi Biotech) and cultured in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 20% FBS, 1× Glutamax (Gibco), and 1 mM HEPES (Gibco). Unless otherwise noted, cells were cultured in 5 ng/ml recombinant IL-2 (Peprotech). After thaw, cells were counted and cultured at 1 million/ml in flat bottom culture plates.
9
Isolation and Characterization of Immune Cells
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Blood was provided from consenting, healthy donors in accordance with the Ethics Committee of Kikkoman Corporation (Chiba, Japan), and PBMCs were isolated by Ficoll-Paque PLUS (GE Healthcare). mDC1 were isolated from PBMCs by CD1c+ (BDCA1+) Dendritic Cell Isolation Kit (Miltenyi Biotec). Cell purity was >98% as assessed by staining with FITC-conjugated anti-CD11c antibody (Ab), BV421-conjugated anti-CD1c Ab and APC-conjugated anti-HLA-DR Ab (BioLegend). Naïve CD4+ T cells were isolated by Naïve CD4+ T Cell Isolation Kit II (Miltenyi Biotec). Cell purity was >98% as assessed by staining with FITC-conjugated anti-CD45RA Ab and APC-conjugated anti-CD4 Ab (BioLegend). Monocyte-derived DCs (moDCs) were prepared by culturing CD14+ monocytes isolated from PBMCs using CD14 Microbeads (Miltenyi Biotec) for 7 days in culture medium including IL-4 and GM-CSF (PeproTech).
10
Naive CD4+ T Cell Polarization
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Untouched naïve human CD4+ T cells were isolated from PMBCs by magnetic-activated cell sorting using the Naïve CD4+ T Cell Isolation Kit II (Miltenyi) and an LS Column (Miltenyi) according to the manufacturer's protocol. TH1 T cell polarization was induced by using 5 µl Dynabeads Human T-Activator CD3/CD28 (Gibco) with 1×106 cells, 10 ng/ml human IFN-γ (Peprotech), 10 ng/ml human IL-12 (Peprotech) and 1 µg/ml anti-IL-4 (Peprotech). T cells were cultured for 14 days in RPMI medium containing L-glutamine and 25 mM HEPES (Gibco) supplemented with 10% FCS, 1% penicillin-streptomycin (Thermo Fisher Scientific) and 0.02 mM sodium pyruvate (Thermo Fisher Scientific) at 37°C and 5% CO2.
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