Isolated human naive T cells were stimulated for 6 days in 96 well, flat bottomed plates (Corning) using 1 µg/mL of plate-bound anti-CD3 (BioLegend Cat# 317302, RRID:AB_571927) and 2 µg/mL anti-CD28 (BioLegend Cat# 302902, RRID:AB_314304). Cells were seeded at 100,000 naive T cells per well. Th1 cells were generated by addition of 10 ng/mL hIFNγ, 10 ng/mL IL-12 and 100 U/mL hIL-2 (all from Miltenyi). Th17 cells were generated using 10 ng/mL hIL-1β, 10 ng/mL hIL-6, 10 ng/mL hIL-23, 100 U/mL hIL-2, 10 ng/mL TGF-β (all from Miltenyi) with 10 µg/mL anti-IFNγ (BioLegend Cat# 502402, RRID:AB_315223). Cells were incubated at 37 °C, 5% CO2. Cells were cultured in complete IMDM (ThermoFisher, with 10% FCS, Penicillin, streptomycin and glutamine).
Hil 2
Manufactured by Miltenyi Biotec
Sourced in Germany
The HIL-2 is a laboratory instrument designed for the automated isolation and enrichment of specific cell populations from complex biological samples. It utilizes a magnetic bead-based separation technology to selectively capture and separate target cells. The core function of the HIL-2 is to enable efficient and reproducible cell isolation, supporting research and applications in the field of cell biology and immunology.
Automatically generated - may contain errors
Lab products found in correlation
Hil 7, by Miltenyi Biotec (6 mentions)
Hil 15, by Miltenyi Biotec (4 mentions)
Siinfekl peptide, by AnaSpec (3 mentions)
Hil 2, by Thermo Fisher Scientific (3 mentions)
Anti cd3 antibody, by Miltenyi Biotec (2 mentions)
Anti cd28 antibody, by Miltenyi Biotec (2 mentions)
Apc conjugated anti human cd3, by BioLegend (2 mentions)
96 well flat bottomed plate, by Corning (1 mentions)
Anti cd3, by BioLegend (1 mentions)
Anti cd28, by BioLegend (1 mentions)
Tgf β, by Miltenyi Biotec (1 mentions)
Il 12, by Miltenyi Biotec (1 mentions)
H ifnγ, by Miltenyi Biotec (1 mentions)
H il 6, by Miltenyi Biotec (1 mentions)
Anti ifn γ, by BioLegend (1 mentions)
Human ab serum, by MP Biomedicals (1 mentions)
Realtime glo, by Promega (1 mentions)
Celltiter glo, by Promega (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
17 protocols using hil 2
1
Th1 and Th17 Cell Differentiation
2
Assessing SGN-CD70A Cytotoxicity in CTCL and T-ALL
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CTCL and T-ALL cell lines were treated with various concentrations of SGN-CD70A or PBS for 72 hours. The cell proliferation was measured by CellTiter-Glo (Promega, Leiden, Netherlands), and apoptosis was analyzed by the annexin-V/PI assay.
Primary tumor cells from PDX mice were cultured in complete media, RPMI1640 with 20% human AB serum (MP, Solon, OH). For the proliferation assays, primary tumor cells from PDXs were stimulated to grow with CD2/CD3/CD28 activation beads and incubated with hIL-2 (500 U/mL) and hIL-7 (1000 U/mL) (Miltenyi Biotec) at an optimal cell density of 5 to 10 × 105 cells/mL. After 24 or 48 hours of activation, the cells were treated with various concentrations of SGN-CD70A as indicated. The proliferation of primary tumor cells was determined by Real-Time Glo (Promega) at 24, 48, and 72 hours after drug treatment. Apoptosis assays using primary tumor cells were performed as described previously.20 (link) Briefly, primary tumor cells were harvested from the spleens of PDX mice and cultured in a complete medium with 250 U/mL of hIL-2 without activation beads. At 24 hours, cells were treated with various concentrations of SGN-CD70A or ADC-IgG control. After 72 hours of treatment, cells were analyzed by the annexin-V/PI assay.
Primary tumor cells from PDX mice were cultured in complete media, RPMI1640 with 20% human AB serum (MP, Solon, OH). For the proliferation assays, primary tumor cells from PDXs were stimulated to grow with CD2/CD3/CD28 activation beads and incubated with hIL-2 (500 U/mL) and hIL-7 (1000 U/mL) (Miltenyi Biotec) at an optimal cell density of 5 to 10 × 105 cells/mL. After 24 or 48 hours of activation, the cells were treated with various concentrations of SGN-CD70A as indicated. The proliferation of primary tumor cells was determined by Real-Time Glo (Promega) at 24, 48, and 72 hours after drug treatment. Apoptosis assays using primary tumor cells were performed as described previously.20 (link) Briefly, primary tumor cells were harvested from the spleens of PDX mice and cultured in a complete medium with 250 U/mL of hIL-2 without activation beads. At 24 hours, cells were treated with various concentrations of SGN-CD70A or ADC-IgG control. After 72 hours of treatment, cells were analyzed by the annexin-V/PI assay.
3
Activation of Naive CD4+ T Cells
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Naive CD4+ T cells were plated at 0.5 × 106 cells/well (200 μl/well) in a 96-well round bottom plate and cultured in medium containing 10 ng/ml hIL-2, hIL-7 and hIL-15 (Miltenyi). Additionally, monoclonal antibodies against CD3 (0.1 μg/ml, clone CLB-T3/4.E, Sanquin) and CD28 (0.2 μg/ml, clone CLB-CD28/1, Sanquin) were added to generate activated CD4+ T cells. The supernatant was collected after 48 h of culture and frozen at −20 °C until analysis. A Human IFN-Beta ELISA Kit with high sensitivity (PBL Assay Science) was used according to the manufacturer’s instructions.
4
Generation and Characterization of Gimap5 Knockout Mice
5
Cytokine-Driven CD8+ T Cell Memory
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OT-1 splenocytes were activated with 20 µg/ml OVA protein (Invivogen) and 100 U/ml hIL-2 (Miltenyi Biotec) and cultured in IMDM media (supplemented with 10% FBS, 2 mM L-glutamine, 1 mM Sodium pyruvate, 0.1 mM NEAA, 1% Kanamycin) for 3 days. After washing, cells were subsequently cultured with 0.4, 2, 10 ng/ml of hIL-15 (R&D) and/or 2 mM MET for 4 days. IL-15-derived antigen-specific CD8+ T cells were analyzed for memory markers by flow cytometry.
6
Isolation and Expansion of CD8+ T Cells
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The peripheral blood of healthy persons (volunteers in our laboratory) was put into anticoagulant tubes. After 1:1 dilution with normal saline, the blood was slowly spread on the surface of the appropriate Ficoll-Paque separation medium (GE). Centrifugation was performed at 800 × g for 20 min. The white film was carefully absorbed after stratification. Centrifugation at 800 g for 5 min. Precipitation of human peripheral blood mononuclear cells. CD8+ T lymphocytes were isolated from human peripheral blood monocytes with a magnetic bead sorting kit (Miltenyi). In vitro expansion was carried out in TexMACS medium (Miltenyi) containing hIL2 (50 U/mL, Miltenyi) and hIL-7-FC (70 ng/mL, Miltenyi) in a 5% CO2 incubator at 37 °C. For the specific steps, please refer to the previous publication of our research group [12 (link)]. KYSE150 cells were conventionally cultured in RPMI 1640 medium (containing 10% FBS, Gibco). When the confluence was 60–70%, CD8+ T lymphocytes were added to the cell culture medium at a ratio of 10:1 CD8+ T lymphocytes: KYSE150 cells and cocultured at 37 °C in a 5% CO2 incubator.
7
Activation of Naive CD4+ T Cells
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Naive CD4+ T cells were flow sorted on the basis of the CD3+HLA-DR-CD4+CD25-/lowCD45RA+ phenotype and cultured for 48-72 h after plating at 0.5 × 106 cells/well in 96-well round bottom plates (BD Falcon) in RPMI-1640 medium supplemented with 10% FBS and Antibiotic–Antimycotic Solution (Sigma) in the presence of hIL-2, hIL-7 and hIL-15 (Miltenyi, each 10 ng/ml); additionally, monoclonal antibodies against CD3 and CD28 were added to activate CD4+ T cells. In addition, CD4+ T cells were treated with a STING inhibitor (H-151, 15 ng/ml; InvivoGen) or a STING agonist (2′3′-cGAMP, 15 μg/ml; InvivoGen) for the last 8 h of culture. Intracellular IFNβ and phosphoprotein detection was performed using flow cytometry.
8
Antigen-specific T Cell Expansion Protocol
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Splenocytes were stimulated with various doses of soluble SIINFEKL peptide (0.1 to 1000 nM) and incubated at 37°C in 10% FCS, 5 mg/mL penicillin, 5 mg/mL streptomycin, 0.05 mM 2‐ME, 10 mM HEPES, and 1 mM sodium pyruvate (all Invitrogen, CA, USA) in RPMI (cRPMI). Cells were split on day 4, following peptide stimulation and 20 U/mL of hIL‐2 (Roche) was added to each well. Long‐term cultures were split every other day, starting from day 2, into cRPMI with 10 U/mL of hIL‐2, 10 ng/mL of hIL‐7 (Miltenyi Biotec, Bergisch Gladbach, Germany) and 10 ng/mL of hIL‐15 (Peprotech, Rehovot, Israel). Unstimulated cells were cultured in the same conditions without peptide and were used as controls.
9
Isolation and Activation of Human T Cells
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Whole blood was taken from healthy donors. Using Ficoll–Paque density gradient centrifugation, human peripheral blood mononuclear cells (PBMCs) were isolated. Isolated PBMCs were seeded into 24-well plates (1.5 × 106 cells/well) and cultured in RPMI1640 containing 10%FBS and 100 IU hIL-2 (Miltenyi Biotec). To activate and enrich T cells, PBMCs were cultured with 3 ug/ml anti-CD3 antibody (Miltenyi Biotec) and 10 ug/ml anti-CD28 antibody (Miltenyi Biotec). After 4 days of incubation at 37 °C, the purity of T cells was assayed using APC conjugated anti-human CD3 (BioLegend, USA) by flow cytometry. The clone of APC anti-human CD3 Antibody was UCHT1.The purity of T cells is represented in the figure S 2 .
10
Murine T Cell Activation and Expansion
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Murine T cells were activated for 24 h with (i) αCD3/CD28 Ab-coated beads (Gibco, Thermo Fisher Scientific) at a bead to cell ratio of 2:1 and 50 IU/ml of hIL-2 (Glaxo), (ii) plate-coated αCD3 Ab (5 µg/ml; eBioscience) plus soluble αCD28 Ab (2 µg/ml; eBioscience) and hIL-2 (50 IU/ml), or (iii) Concanavalin A (2 µg/ml; Sigma), hIL-7 (1 ng/ml; Miltenyi) and 50 IU/ml hIL-2 before transduction. Transduced T cells were cultured at a concentration of 0.5–106 cells/ml in T cell medium enriched with 50 IU/ml hIL-2 only or with 10 ng/ml of both hIL-7 and hIL-15 (Miltenyi) from day 2 after transduction onwards. T cells were typically counted every 2–3 d. T cell expansion was calculated by dividing the absolute number of expanded T cells at each time point during culture by the respective number on day 0 (T cell transduction). T cell viability was assessed by trypan blue staining, and the phenotype was assessed by cell-surface staining of CD44 and CD62L followed by flow cytometric analysis.
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