Human samples were obtained through an institutional review board approved protocol. Blood was drawn from a total of 8 healthy volunteers in 3 independent experiments. PBMCs were extracted after Ficoll separation during centrifugation (Ficoll-Paque PLUS, GE Healthcare). CD4+ T cells were purified by magnetic bead separation according to the manufacture’s instructions (Human CD4+ T cell enrichment kit, Stem Cell Technologies). Purified CD4+ T cells were stimulated 20 hrs with dynabeads human T- activator anti-CD3/anti-CD28 beads (Gibco). The 15% biggest ‘mTORhi’ and smallest ‘mTORlo’ CD38+, CD4+ T cells were sorted on a BD FACS Aria II. Sorted cells were cultured in media supplemented with 3,000units/ml human IL-2 (provided by the NCI) for 72 hrs. Phenotype of sorted cells was determined by flow cytometry.
Human cd4 t cell enrichment kit
Manufactured by STEMCELL
Sourced in Canada
The Human CD4+ T Cell Enrichment Kit is a laboratory product that allows for the isolation and purification of CD4+ T cells from human blood or tissue samples. The kit uses magnetic beads coated with antibodies to selectively bind and separate the CD4+ T cells from the rest of the cell population.
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Lab products found in correlation
Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (4 mentions)
Ficoll paque plus, by GE Healthcare (3 mentions)
Facsaria 2, by BD (2 mentions)
Human naive cd8 t cell isolation kit, by STEMCELL (2 mentions)
Anti cd45ro magnetic beads, by Miltenyi Biotec (2 mentions)
Anti cd14 magnetic bead, by Miltenyi Biotec (2 mentions)
Lymphoprep, by STEMCELL (2 mentions)
Anti cd69, by STEMCELL (2 mentions)
Facsaria, by BD (2 mentions)
Lymphocyte separation medium, by Corning (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Recombinant cytokines, by R&D Systems (1 mentions)
Complete rpmi 1640 medium, by Euroclone (1 mentions)
Human cd4 cd25 cd127dim regulatory t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
Facscalibur cytometer, by BD (1 mentions)
Human memory cd4 t cell enrichment kit, by STEMCELL (1 mentions)
Human cd28 microbead kit, by Miltenyi Biotec (1 mentions)
Pan monocyte microbeads, by Miltenyi Biotec (1 mentions)
Bicoll separating solution, by Harvard Bioscience (1 mentions)
26 protocols using human cd4 t cell enrichment kit
1
Isolation and Expansion of mTOR-Regulated CD4+ T Cells
2
Activation and Cytokine Treatment of CD4 T Cells
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CD4 T cells were negatively selected from PBMCs using the human CD4 T cell enrichment kit (Stem cell Technologies) to provide >98% CD4 T cells (data not shown). The cells were activated for 5 days with bead-bound anti-TCR/CD28 antibodies in the presence of IL-2. This method results in cells that are 100% positive for CD4 and 90–96% positive for CD45RO [18 (link)]. The cells were rested without stimulation for 24 h in complete RPMI (RPMI 1640 with 10% FBS, penicillin/streptomycin, and l-glutamine). The activated CD4 T cells were then treated with or without recombinant cytokines (R&D Systems) for different times and doses in complete RPMI. After pretreatment, cells were washed with RPMI 1640 three times to remove the cytokines.
3
Isolation and Purification of Immune Cell Subsets
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For the in vitro experiments, whole blood samples from healthy donors were collected in EDTA-tubes by the Department of Transfusion Medicine (University Hospital of Udine) after a written informed consent was signed.
PBMCs were separated by centrifugation at 700 g for 20 minutes on a Ficoll Hypaque density gradient (Millipore) and resuspended at 1 x 106 cells/ml in RPMI 1640 complete medium supplemented with 10% uFBS, 1% glutamine, 1% Na pyruvate, 1% non-essential aminoacid, 1% penicillin/streptomycin, 1% Hepes and 50μM β-mercapthoethanol (all from Euroclone).
CD4+ T cells were purified from PBMCs by negative selection using a human CD4+ T cell enrichment kit (StemCell Technologies), according to the manufacturer’s instructions. Purity of monocytes was over 95% as proved by staining with anti-CD14 mAb (eBiosciences) and flow cytometry analysis (FACScalibur cytometer, BD, Biosciences).
Treg cells were isolated from PBMC with human CD4+CD25+CD127dim/- Regulatory T cell isolation kit II (Miltenyi Biotec). Purification efficiency was over 95%.
To remove monocytes, PBMC were stained with an anti-CD14 fluorescent antibody (clone 61D3, PE conjugated, eBiosciences) and CD14 negative cells were sorted by FACSAria II cell sorter (BD Biosciences). As a control, unfractioned PBMCs were sorted as well.
PBMCs were separated by centrifugation at 700 g for 20 minutes on a Ficoll Hypaque density gradient (Millipore) and resuspended at 1 x 106 cells/ml in RPMI 1640 complete medium supplemented with 10% uFBS, 1% glutamine, 1% Na pyruvate, 1% non-essential aminoacid, 1% penicillin/streptomycin, 1% Hepes and 50μM β-mercapthoethanol (all from Euroclone).
CD4+ T cells were purified from PBMCs by negative selection using a human CD4+ T cell enrichment kit (StemCell Technologies), according to the manufacturer’s instructions. Purity of monocytes was over 95% as proved by staining with anti-CD14 mAb (eBiosciences) and flow cytometry analysis (FACScalibur cytometer, BD, Biosciences).
Treg cells were isolated from PBMC with human CD4+CD25+CD127dim/- Regulatory T cell isolation kit II (Miltenyi Biotec). Purification efficiency was over 95%.
To remove monocytes, PBMC were stained with an anti-CD14 fluorescent antibody (clone 61D3, PE conjugated, eBiosciences) and CD14 negative cells were sorted by FACSAria II cell sorter (BD Biosciences). As a control, unfractioned PBMCs were sorted as well.
4
Isolation and Separation of Blood Cells
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The study protocols were approved by the institutional review board of Seoul National University Hospital and Severance Hospital. Peripheral blood of ESRD patients and healthy controls (HCs) was drawn after obtaining written, informed consent. The methods were performed in accordance with the approved guidelines. Peripheral blood mononuclear cells (PBMC) were isolated from blood by density gradient centrifugation (Bicoll separating solution; BIOCHROM, Cambridge, UK). Total monocytes were negatively separated from PBMC with pan-monocyte microbeads (Miltenyi Biotec, Auburn, CA), if no special mention in the figure legend. Total CD4+ T cells and CD4+ memory T cells were negatively enriched from PBMC using human CD4 T cell enrichment kit and human memory CD4 T cell enrichment kit (STEMCELL Technologies, Vancouver, Canada), respectively. CD4+CD28+ and CD4+CD28− T cells were separated from CD4+ memory T cells using a human CD28 microbead kit (Miltenyi Biotec) as described in user’s instructions. CD28− cells were retained in the run-through fraction including CD28 unlabeled cells.
5
Purification and Activation of CD4+ T Cells
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CD4+ T lymphocytes were purified from PBMCs isolated from three anonymous blood donors (with ages ranging from 18 to 65 years) by negative selection using the Human CD4+ T Cell Enrichment Kit (#19052, Stemcell Technologies) according to the manufacturers' instructions. Purification was over 90% as assessed by staining with 5 ng/μl of anti-CD4-FITC (clone RPA-T4, eBioscience) and flow cytometric analysis (FACSCalibur).
Isolated CD4+ T lymphocytes (2 × 105) were resuspended in RPMI medium (control) or preincubated for 24 hours with 50% of AMSC-derived CM and SF, then seeded in triplicate into 96-well plates with prebound 0.5 μg/ml anti-CD3 (clone OKT3, eBiosciences), 0.5 μg/ml anti-CD28 (clone CD28.6, eBiosciences), and recombinant IL-2 at a concentration of 250 U/ml (Peprotech). After 3 days, in vitro-stimulated CD4+ T cells were stained with 5 ng/μl of anti-CD25-APC (clone BC96, eBiosciences) and 5 ng/μl of anti-FoxP3-PE (clone PCH101, eBioscience) and T reg proliferation was tested with flow cytometry (FACSCalibur, Becton Dickinson). The data were analysed using the Flowing Software.
Isolated CD4+ T lymphocytes (2 × 105) were resuspended in RPMI medium (control) or preincubated for 24 hours with 50% of AMSC-derived CM and SF, then seeded in triplicate into 96-well plates with prebound 0.5 μg/ml anti-CD3 (clone OKT3, eBiosciences), 0.5 μg/ml anti-CD28 (clone CD28.6, eBiosciences), and recombinant IL-2 at a concentration of 250 U/ml (Peprotech). After 3 days, in vitro-stimulated CD4+ T cells were stained with 5 ng/μl of anti-CD25-APC (clone BC96, eBiosciences) and 5 ng/μl of anti-FoxP3-PE (clone PCH101, eBioscience) and T reg proliferation was tested with flow cytometry (FACSCalibur, Becton Dickinson). The data were analysed using the Flowing Software.
6
Immune Cell Isolation from Healthy Donors
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Peripheral blood samples were obtained from 27 female and 12 male healthy individuals who did not have a history of autoimmune disease, diabetes mellitus, renal disease, cardiovascular disease or cancer except skin cancer. Individuals with hypertension or hypercholesterinemia were included if controlled on treatment. 19 of these 39 individuals were older than 60 years. In addition, samples were obtained from 128 blood or platelet donors. These samples were deidentified except for whether donors were younger than 35 years or older than 60 years. The studies were approved by the Stanford University Institutional Review Board, and participants gave informed written consent.
Untouched CD4+ T cells were purified from peripheral blood or leukapheresis samples of healthy volunteers with a human CD4+ T Cell enrichment kit (STEMCELL Technologies), followed by density gradient centrifugation using Lymphoprep (STEMCELL Technologies). Naive CD4+ T cells were further isolated by negative selection with anti-CD45RO magnetic beads (Miltenyi Biotec). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. Naive CD8+ T cell were isolated from PBMCs using a human naive CD8+ T cell isolation kit (STEMCELL Technologies). CD14+ monocytes were isolated from PBMCs using anti-CD14 magnetic beads (Miltenyi Biotec). Purity of isolated cells was 90% or higher.
Untouched CD4+ T cells were purified from peripheral blood or leukapheresis samples of healthy volunteers with a human CD4+ T Cell enrichment kit (STEMCELL Technologies), followed by density gradient centrifugation using Lymphoprep (STEMCELL Technologies). Naive CD4+ T cells were further isolated by negative selection with anti-CD45RO magnetic beads (Miltenyi Biotec). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. Naive CD8+ T cell were isolated from PBMCs using a human naive CD8+ T cell isolation kit (STEMCELL Technologies). CD14+ monocytes were isolated from PBMCs using anti-CD14 magnetic beads (Miltenyi Biotec). Purity of isolated cells was 90% or higher.
7
Quantifying HIV Latency Activation
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We collected 25 ml of total blood from HIV-infected individuals participating in the Swiss HIV Cohort Study (http://www.shcs.ch ) with controlled viremia (Table S1
8
Isolation and Sorting of T Regulatory Cells
9
Isolation and Activation of CD4+ T Cells
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Peripheral blood samples were obtained after written consent from the donor. Following isolation of peripheral blood mononuclear cells (PBMCs) by Ficoll-Hypaque density gradient centrifugation, CD4+ T cells were isolated by negative selection with the human CD4+ T Cell Enrichment Kit (Stem Cell Technologies Inc., Vancouver, Canada). Purity of the CD4+ cell population was higher than 93% as measured by flow cytometry. Cells were cultured at a density of 2 × 106 cells per ml in RPMI-1640 medium supplemented with 10% FBS, L-glutamine (2 mM) and antibiotics (50 U/ml penicillin, 50 μg/ml streptomycin and 100 μg/ml primocin). For CD4+ T cell activation, PHA-L (1 μg/ml) (Sigma-Aldrich) and recombinant human IL-2 (3000 U/ml) (Feldan-bio, Montreal, Canada) were added to the culture medium for 24 h prior to virus infection.
10
Isolation and Sorting of CD4+ T Cell Subsets
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