T cells were kept at a density of ∼106 cells per ml cell culture medium. The T-cell medium consisted of RPMI 1640 (Invitrogen), 1× penincillin/streptomycin (Invitrogen), 5% FCS, 5% human serum, 1 mM sodium pyruvate (Invitrogen), 2 mM L-glutamine (Invitrogen), 10 mM nonessential amino acids (Invitrogen), 10 mM Hepes (Invitrogen), and 16 μg/ml gentamycin (Biochrom). Human IL-7 and human IL-15 (both PeproTech) were added to the medium to a final concentration of 5 ng/ml each and replenished when fresh culture medium was added to the cells every 2–3 d. The AML cell line ML2 (The CABRI consortium) was retrovirally transduced with genes encoding firefly luciferase (fluc) or HLA-B7. Mycoplasma contamination status was regularly tested.
Human il 7
Manufactured by Thermo Fisher Scientific
Sourced in United States
Human IL-7 is a recombinant protein that represents the human interleukin-7 cytokine. Interleukin-7 is a key regulator of T cell development and homeostasis. It plays a crucial role in the survival and proliferation of T cells.
Automatically generated - may contain errors
Lab products found in correlation
Human il 15, by Thermo Fisher Scientific (5 mentions)
Il 15, by Thermo Fisher Scientific (4 mentions)
Pcmv dr8, by Addgene (3 mentions)
Pcmv vsv g, by Addgene (3 mentions)
Pan t cell isolation kit, by Miltenyi Biotec (2 mentions)
Human il 2, by Thermo Fisher Scientific (2 mentions)
Multisizer 3 coulter counter, by Beckman Coulter (2 mentions)
Cts dynabeads cd3 cd28, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Gentamycin, by Harvard Bioscience (1 mentions)
Easysep human t cell isolation kit, by STEMCELL (1 mentions)
Cd8 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Cd14 microbead, by Miltenyi Biotec (1 mentions)
Cellxvivo human monocyte derived dc differentiation kit, by R&D Systems (1 mentions)
X vivo, by Thermo Fisher Scientific (1 mentions)
Nucleospin tissue, by Takara Bio (1 mentions)
15 protocols using human il 7
1
Expansion and Maintenance of T Cells
2
Lentivirus Production and T Cell Transduction
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Example 3
Lentivirus was produced by transiently transfecting HEK 293T cells using calcium phosphate. Briefly, 10 μg of transfer gene, 7.5 μg of pCMV-dR8.2 (Addgene) and 5 μg of pCMV-VSVG (Addgene) were mixed and incubated with 2 M CaCl2 followed by 2×HBSS. Resulting solutions were added dropwise to 10 cm2 cell culture dishes seeded with 3.2×106 HEK 293T cells in 10 ml DMEM 24h previously. Transfection media was replaced after 6 h. Media containing lentivirus was harvested at 48 and 72 h post transfection, filtered through 0.45 μm filters, and concentrated by ultracentrifugation at 75,000×g for 2 h at 4° C. Lentivirus was then resuspended in serum containing media and frozen at −80° C. Human T cells were transduced 24-72 h post activation with anti-CD3/CD28 Dynabeads either by spinfection (1,000 g for 1 h at 32° C.) or by overnight incubation with lentivirus. T cells were transduced once more 24 h after the first transduction. During and following transductions, media containing IL-2 was replaced with media containing human IL-7 (10 ng/ml) and IL-15 (5 ng/ml) (Peprotech). Jurkat T cells were transduced by a single overnight incubation with lentivirus.
3
Isolation and Coculture of CD8+ T Cells
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Human CD14+ cells and T cells were isolated from cryopreserved PBMCs using CD14 MicroBeads (Miltenyi Biotec) and EasySep Human T Cell Isolation Kit (STEMCELL Technologies), respectively. Monocyte DCs were generated from human CD14+ cells using CellXVivo Human Monocyte-Derived DC Differentiation Kit (R&D Systems). Immature DCs were incubated with or without either GBM6-AD lysate (10 μg/mL) or recombinant human EphA2 and IL-13Rα2 (10 μg/mL, Sino Biological) for 8 hours. DCs and T cells were cocultured at the ratio of 1:4 in X-VIVO supplemented with 2% human AB serum and 5 ng/mL of human IL-7 (PeproTech) for 5 days. CD8+ T cells were isolated from the cocultured cells using the CD8+ T-Cell Isolation Kit (Miltenyi Biotec). Genomic DNA was extracted from CD8+ T cells using NucleoSpin Tissue (Takara Bio) for bulk TCR-Seq.
4
T cell priming by activated Mo-DCs
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T cells were enriched by either recovery of non-adherent cells after 2 h of PBMCs plastic adherence or by negative magnetic selection with the Pan T cell isolation kit (Milteny Biotec). Aliquots of T cells were cryopreserved until used in subsequent experiments. For T cell priming, activated Mo-DCs were harvest, washed, counted, and co-cultured for 7 days with autologous T cells at a 1:10 ratio in 96-well U-bottom plates (Corning, Tewksbury, MA, USA), with human IL-7 (50ng/ml), IL-2 (250 U/ml), and IL-15 (5 ng/ml) (all from Peprotech) with media replacement with fresh cytokines every 3 days. T cell proliferation was determined by carboxyfluorescein succinimidyl ester (CellTrace™ CFSE Cell Proliferation Kit, #C34554, Thermo Fisher Scientific) dilution, where T cells were previously stained with 5 μM CFSE. After 7 days, T cells were harvested and stained for surface markers CD3 (SK7, #340542, BD), PD1 (EH12.1, #561273, BD), TIM3 (F38-2E2, #25-3109-42, Thermo Fisher Scientific), LAG3 (3DS223H, #12-2239-41, Thermo Fisher Scientific), and CFSE dilution was determined by flow cytometry.
5
Enrichment and Transduction of Mouse T Cells
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Mouse spleens were removed from euthanized C57BL/6J mice, and mouse T cells were enriched by using a mouse pan-T Isolation Kit (Miltenyi Biotec, Germany). Isolated T cells were incubated in medium supplemented with human IL-2 (10 ng/ml, PeproTech, Rocky Hill, USA) and human IL-7 (2 ng/ml, PeproTech, Rocky Hill, USA) and were cocultured with mouse T cell activator beads (Miltenyi Biotec, Germany) for 48 h and then transduced with retrovirus by centrifuging at 1,200g for 90 min and then placed in a cell culture incubator (37°C, 5% CO2) for 24 h. CAR-T cells determined by flow cytometry at day 5, as GFP+, and then were included in the experiments.
6
Notch Signaling Activation in Cultured Human T Cells
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Human T cells were activated using the methods mentioned above. To activate Notch signaling, activated T cells were cocultured with OP9‐hDLL1 cells. Human T cells and OP9‐hDLL1 cells were cocultured with human IL‐2 (20 ng/mL; PeproTech), human IL‐7 (10 ng/mL; PeproTech), human IL‐15 (20 ng/mL; Biolegend), or human IL‐21 (20 ng/mL; PeproTech) in Minimum Essential Medium Eagle‐alpha modification for 11 days.
7
Generation and Transduction of Anti-Mesothelin CAR T Cells
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The anti-mesothelin M5 4-1BB-zeta CAR was previously described (25 (link), 29 (link), 30 ). CD4 and CD8 T cells were combined at a 1:1 ratio and rested overnight with R10 media supplemented with 5ng/mL human IL7 and 5ng/mL human IL15 (Preprotech). The next day, CRISPR-Cas9 editing was performed, and cells were cultured in cytokine-supplemented R10 media at 5 × 106 cells/mL at 37 °C and activated 4 to 6 h later with Dynabeads® CD3/CD28 CTS™ (Thermofisher) at a 3:1 bead-to-cell ratio at 1 × 106 cells/mL. After 24 h, T cells were transduced at a multiplicity of infection (MOI) of 3. At day 5, beads were removed from cultures. T cell cultures were maintained at 6 × 105 cells/mL. Cell number and volume were monitored daily using Multisizer 3 Coulter Counter (Beckman). Transduced T cells were cryopreserved when they reached a rested state, as determined by cell volume.
8
Activation and Expansion of Primary Human T Cells
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Peripheral blood mononuclear cells (PBMCs) were collected from healthy voluntary donors and isolated from the whole blood via Ficoll-Paque PLUS density gradient centrifugation (cat# 17144002, cytiva). CD3+ T cells were isolated using the Pan T cell isolation kit (cat# 130-096-535, Miltenyi Biotec). Next, the isolated CD3+ T cells were stimulated for 48 h with the human T cell activation/expansion kit (cat# 130-091-441, Miltenyi Biotec) in accordance with the manufacturer’s instructions. For this process, the T cells were cultured in X-VIVO™ 15 media (cat# 02-060Q, Lonza) supplemented with 10% inactivated FBS, 10 ng/mL human IL-7 (cat# 200-07, Peprotech), and 10 ng/mL human IL-15 (cat# 200-15, Peprotech) at a final concentration of 1 × 106 cells/mL.
9
Isolation and Activation of Human T Cells
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Peripheral blood was obtained from the healthy donors. Blood sampling was performed following the required ethical procedures. Lymphocytes were isolated by density gradient centrifugation following the manual of the Human Lymphocyte Separation Medium (DAKEWE). Human T cells were purified using the human CD3 T cell isolation kit (BioLegend), stimulated with CD3/CD28 T cell Activator Dynabeads (Gibco) and cultured at 106/mL in X-VIVO 15 Serum-free Hematopoietic Cell Medium (Lonza), supplemented with 5 ng/mL human IL-7 (PeproTech) and 5 ng/mL human IL-15 (PeproTech)40 (link),65 (link), with slight modification. 48 h after T cell stimulation, T cells were transduced with lentiviral supernatants from 293 T cell line in the presence of polybrene (6 μg/mL, Yeasen) by centrifugal infection. T cells were analyzed by flow cytometry 4 days after transduction and were used for further experiments. All human subjects were informed and signed informed consent prior to inclusion in the study and all human cell isolation and related experiments were approved by the Ethics Committee for Human Studies of Second Affiliated Hospital of Zhejiang University School of Medicine (2019 NO.388).
10
Lentivirus Production and T Cell Transduction
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Example 3
Lentivirus was produced by transiently transfecting HEK 293T cells using calcium phosphate. Briefly, 10 μg of transfer gene, 7.5 μg of pCMV-dR8.2 (Addgene) and 5 μg of pCMV-VSVG (Addgene) were mixed and incubated with 2 M CaCl2 followed by 2×HBSS. Resulting solutions were added dropwise to 10 cm2 cell culture dishes seeded with 3.2×106 HEK 293T cells in 10 ml DMEM 24 h previously. Transfection media was replaced after 6 h. Media containing lentivirus was harvested at 48 and 72 h post transfection, filtered through 0.45 μm filters, and concentrated by ultracentrifugation at 75,000×g for 2 h at 4° C. Lentivirus was then resuspended in serum containing media and frozen at −80° C. Human T cells were transduced 24-72 h post activation with anti-CD3/CD28 Dynabeads either by spinfection (1,000 g for 1 h at 32° C.) or by overnight incubation with lentivirus. T cells were transduced once more 24 h after the first transduction. During and following transductions, media containing IL-2 was replaced with media containing human IL-7 (10 ng/ml) and IL-15 (5 ng/ml) (Peprotech). Jurkat T cells were transduced by a single overnight incubation with lentivirus.
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