CD4+ T cells were isolated by negative selection from freshly obtained PBMC using human CD4+ T cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany). Purity was confirmed with CD4-FITC (Cat# 555346, BD Bioscience) and analyzed by flow cytometry. CD8+ T cells were isolated by negative selection from freshly obtained PBMC using human CD8+ T cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany). Purity was confirmed with CD8-FITC (Cat# 555366, BD Bioscience) and analyzed by flow cytometry. Cells were cultured in RPMI 1640 medium (Sigma) supplemented with 10% heat-inactivated endotoxin-tested FCS (Biochrom GmbH, Berlin, Germany).
Human cd4 t cell isolation kit
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
The Human CD4+ T cell isolation kit is a lab equipment product that is used to isolate CD4+ T cells from human samples. It utilizes magnetic cell separation technology to selectively enrich for CD4+ T cells.
Automatically generated - may contain errors
Lab products found in correlation
Human cd8 t cell isolation kit, by Miltenyi Biotec (8 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (7 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (4 mentions)
Facsaria 2, by BD (3 mentions)
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (3 mentions)
Fitc mouse anti human cd4, by BD (2 mentions)
Ficoll hypaque solution, by Cedarlane (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Automacs, by Miltenyi Biotec (2 mentions)
Cd14 microbead, by Miltenyi Biotec (2 mentions)
Whole human genome dna microarray 4 44k, by Agilent Technologies (2 mentions)
Human monocyte isolation kit 2, by Miltenyi Biotec (2 mentions)
Human pan monocyte isolation kit, by Miltenyi Biotec (2 mentions)
Human cd14 microbeads, by Miltenyi Biotec (2 mentions)
Facsaria, by BD (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Ficoll paque, by GE Healthcare (2 mentions)
Cd25 microbeads, by Miltenyi Biotec (2 mentions)
Mouse cd4 t cell isolation kit, by Miltenyi Biotec (2 mentions)
71 protocols using human cd4 t cell isolation kit
1
Isolating CD4+ and CD8+ T Cells
2
Isolation and Analysis of CD4+ T Cells
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Human fresh peripheral blood samples of all individuals were obtained from the children with acute HSP and HCs. Peripheral blood mononuclear cells (PBMCs) isolated by density gradient centrifugation with Ficoll-Hypaque solution (CL5020, CEDARLANE, Netherlands) were transferred to sterile tubes and washed twice with phosphate-buffered saline (PBS). Subsequently, human CD4+T cells were isolated by human CD4+T cell isolation kit (number 130-096-533) (Miltenyi Biotec GmbH, Germany). Additionally, human PBMCs were stained with FITC-Mouse Anti-Human CD4 (BD Biosciences, San Diego, CA, USA), clone name: RPA-T4, isotype control: FITC Mouse IgG2b, κ, clone name: eBMG2b; PE-Mouse Anti-Human CXCR5 Biotinylated Monoclonal Antibody (BioLegend, San Diego, CA), clone name: J252D4, isotype control: PE Mouse IgG2a, κ, clone name: eBM2a; APC-Anti-Human ICOS (CD278) (BD Biosciences, San Diego, CA, USA), clone name: ISA-3, isotype control: APC Mouse IgG2a, κ, clone name: MOPC-173; PerCP-Cy5.5 Mouse Anti-Human CD279 (PD-1) (BD Biosciences, San Diego, CA, USA), clone name: EH12.1, isotype control: PerCP-Cy5.5 Mouse IgG1, κ, clone name: MOPC-21. All the staining procedures were performed according to the manufacturers' protocols, and the stained cells were analyzed by a flow cytometer of FACSCalibur and CELLQUEST software (Becton Dickinson, Sparks, MD, USA).
3
Activation and Transduction of Primary Human CD4+ T Cells
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Human primary CD4+ T cells were obtained from peripheral blood mononuclear cells (PBMCs) from healthy volunteer donors through the Infectious Diseases BioBank at King’s College London (ethics reference MM2-220518) under overall permission from the Southampton and South West Hampshire Research Ethics Committee (REC; B) (REC reference 19/SC/0232). PBMCs were isolated using density gradient centrifugation in SepMate tubes (STEMCELL Technologies) with Lymphoprep density gradient medium (STEMCELL Technologies). Total CD4+ T cells were isolated using a Human CD4+ T Cell Isolation Kit (Miltenyi Biotec). CD4+ T cells were cultured in RPMI-1640 medium with GlutaMAX and HEPES supplemented with 10% heat-inactivated autologous human serum and 1% penicillin-streptomycin (Life Technologies). Cells were then activated using Dynabeads Human T-Activator CD3/CD28 (Life Technologies) and recombinant human interleukin-2 (30 U/mL; Roche) for 48 h prior to infection.
Activated CD4+ T cells were transduced with lentiviral vectors (1−10 ng of p24Gag of vector per 50,000 cells). 48 h post-transduction, the cells were fixed in 4% paraformaldehyde in DPBS before assessing transduction efficiency by flow cytometry measuring intracellular GFP expression.
Activated CD4+ T cells were transduced with lentiviral vectors (1−10 ng of p24Gag of vector per 50,000 cells). 48 h post-transduction, the cells were fixed in 4% paraformaldehyde in DPBS before assessing transduction efficiency by flow cytometry measuring intracellular GFP expression.
4
Isolation of CD4+ and CD8+ T Cells
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CD4+T and CD8+T cells were isolated by human CD4+ T Cell Isolation Kit or human CD8+ T Cell Isolation Kit following the protocol from Miltenyi biotec using MidiMACS.
5
CD4+ T Cell Transcriptome Analysis
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CD4+ T cells were separated from PBMCs using the Human CD4+ T cell Isolation Kit (Miltenyi Biotech KK., Tokyo, JAPAN). Total RNA from these cells was extracted. After quality checking by an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, U.S.A.), 50 μg of total RNA was pre-amplified and labeled with cyanine 3-CTP. Complementary DNA was hybridized onto the G4112F Whole Human Genome cDNA microarray 44K × 4plex ver.1.0 (Agilent Technologies). After hybridization, the raw images were acquired and converted into fluorescence intensity data. The data were imported to GeneSpring GX Software ver. 7.3.1 (Agilent Technologies).
6
Isolation of CD4+ T Cells from Human PBMCs
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Human peripheral blood samples were obtained from the Department of Transfusion Medicine at the University Hospital Würzburg, and use of the samples complied with the approval of the University Hospital of Würzburg Ethical Committee. PBMCs were isolated by standard Ficoll gradient centrifugation (Bicoll; Biochrom). CD4+ T cells were enriched using a human CD4+ T cell isolation kit (Miltenyi Biotec).
7
Exosome-Mediated Adenosine Metabolism in T Cells
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Healthy donor’s PBMCs were harvested as described above. CD4+ CD39+ T cells were isolated from PBMCs by negative selection of CD4+ cells using the human CD4+ T Cell Isolation Kit (130-096-533) followed by positive selection of CD39+ cells using a biotinylated anti-CD39 antibody (130-093-505, RRID:AB_1036212) and anti-biotin MicroBeads (130-090-485, RRID:AB_244365, all from Miltenyi Biotec) according to manufacturer’s instruction and as described previously [13 (link), 22 (link)].
Freshly isolated CD4+ CD39+ T cells (25,000 cells in 50 µl PBS) were incubated with exosomes (10 µg in 50 µl PBS) isolated from plasma of HNSCC patients and 20 µM adenosine triphosphate (ATP) for 1 h at 37 °C. Additionally, exosomes alone (10 µg in 100 µl PBS) were incubated with 20 µM ATP for 1 h at 37 °C. Samples were centrifuged at 6000 × g for 2 min, supernatants were boiled for 2 min at 95 °C and stored at −80 °C until further processing. Concentrations of 5‘ adenosine monophosphate (AMP) and adenosine, as well as their degradation product hypoxanthine, were measured by mass spectrometry, as described previously [22 (link)]. As controls, T cells incubated with ATP, exosomes incubated without ATP, and ATP in PBS only were used.
Freshly isolated CD4+ CD39+ T cells (25,000 cells in 50 µl PBS) were incubated with exosomes (10 µg in 50 µl PBS) isolated from plasma of HNSCC patients and 20 µM adenosine triphosphate (ATP) for 1 h at 37 °C. Additionally, exosomes alone (10 µg in 100 µl PBS) were incubated with 20 µM ATP for 1 h at 37 °C. Samples were centrifuged at 6000 × g for 2 min, supernatants were boiled for 2 min at 95 °C and stored at −80 °C until further processing. Concentrations of 5‘ adenosine monophosphate (AMP) and adenosine, as well as their degradation product hypoxanthine, were measured by mass spectrometry, as described previously [22 (link)]. As controls, T cells incubated with ATP, exosomes incubated without ATP, and ATP in PBS only were used.
8
Purification and Activation of Immune Cells
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Primary B cells, T cells, and monocytes were purified from PBMCs by positive selection with CD19, CD3, and CD14 magnetic beads, respectively (Miltenyi). For quantitative PCR, CD4+ T cells were purified from PBMCs by negative selection using the human CD4+ T-cell isolation kit (Miltenyi) and were stimulated with 2.5 μg/mL plate-bound anti-CD3 antibody (clone OKT3) and 0.25 μg/mL soluble anti-CD28 antibody for 0, 6, 18, and 24 h. Jurkat cells and B-lymphoblastoid cell lines (B-LCLs) were cultured in RPMI 1640 medium supplemented with 10% FBS (HyClone; Thermo Scientific), 2.05 mmol/L L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, and 1 mmol/L sodium pyruvate (Life Technologies) at 37°C with 5% CO2.
9
Isolation and Culture of CD4+ T Cells
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PBMCs were isolated from blood samples by Ficoll-Hypaque density-gradient centrifugation. CD4+ T cells were enriched from PBMCs by magnetic separation (AutoMACS, Miltenyi Biotec) using human CD4+ T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions before flow sorting on a FACSAria II (BD Biosciences).
CD4+ T cells were activated with anti-CD3/CD28-coated beads (Life Technologies Dynabeads T-Activator) and IL-2 (20 IU/mL) and cultured in RPMI 1640 medium (Life Technologies) supplemented with 1% penicillin-streptomycin (Life Technologies), 2 mM GlutaMAX (Life Technologies), 1 mM sodium pyruvate (STEMCELL Technologies) for the indicated time intervals.
Typical cell culture conditions (37°C, 5% CO2, and environmental [21%] oxygen) were used and the conditions referred to as normoxia. Hypoxia was generated with an oxygen/CO2 controller incubator (Galaxy 48R; Eppendorf). It was set at 1% oxygen and 5% CO2.
CD4+ T cells were activated with anti-CD3/CD28-coated beads (Life Technologies Dynabeads T-Activator) and IL-2 (20 IU/mL) and cultured in RPMI 1640 medium (Life Technologies) supplemented with 1% penicillin-streptomycin (Life Technologies), 2 mM GlutaMAX (Life Technologies), 1 mM sodium pyruvate (STEMCELL Technologies) for the indicated time intervals.
Typical cell culture conditions (37°C, 5% CO2, and environmental [21%] oxygen) were used and the conditions referred to as normoxia. Hypoxia was generated with an oxygen/CO2 controller incubator (Galaxy 48R; Eppendorf). It was set at 1% oxygen and 5% CO2.
10
Isolation and Culture of CD4+ T Cells with NK-EVs
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CD4+ T cells were isolated from non-adherent PBMCs, using the human CD4+ T cell isolation kit (MACS Miltenyi Biotec). Isolated T helper cell purity was checked by flow cytometry CD4 staining and they were then cultured in CD3 (5 µg/ml) and CD28 (2 µg/ml) coated plates either in the presence or absence of NK-EVs and both in non-polarizing (RPMI supplemented with 10% EV-free FBS) and Th1-polarizing conditions, adding a mixture of cytokines (20 U/ml IL-2 and 20 ng/ml IL-12). NK-EVs obtained from 25 × 106 NK cells supernatants, as described above, were resuspended in 100 µl of EXO-free RPMI and 20 µl were added to 5 × 106 CD4+ T cells per condition. On days 3 and 6 post-coculture, cells in the different conditions and their supernatants were analysed as described below. NK-EVs isolated from three different healthy donors were cocultured with Th cells from at least two different donors.
For EV-uptake blockade experiments, isolated CD4+ T cells were supplemented with 80 µM Dynasore (Sigma-Aldrich) before NK-EV incubation.
For EV-uptake blockade experiments, isolated CD4+ T cells were supplemented with 80 µM Dynasore (Sigma-Aldrich) before NK-EV incubation.
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