PBMCs from healthy adult peripheral blood samples were isolated by Ficoll centrifugation. Human PBMCs were plated at 3–5 × 106 cells/mL and preactivated overnight (about 16 h) using rhIL-12 (10 ng/mL, PeproTech) + rhIL-18 (50 ng/mL, MBL International) + rhIL-15 (1 ng/mL, PeproTech) or control (rhIL-15, 1 ng/mL) conditions, washed three times to remove cytokines, and then added with rhIL-15 (1 ng/mL) to maintain the culture in a complete RPMI 1640 medium containing 10% fetal bovine serum. After a week of culture at 37°C in a 5% CO2 incubator, the cells were washed, added with PMA + Ionomycin (cell activation cocktail with brefeldin was obtained from BioLegend (San Diego, CA), the same set of cytokines IL-12/15/18, K562 tumor cell lines, 0.1–1 µM of the CDK4/6 inhibitor, Myc inhibitor, DNA-demethylating agent, and enhancer of zeste homolog2 (EZH2) inhibitor separately for 5 h), and then washed three times with phosphate-buffered saline.
Rhil 15
Manufactured by Thermo Fisher Scientific
Sourced in United States
RhIL-15 is a recombinant human interleukin-15 protein produced in E. coli. Interleukin-15 is a cytokine that plays a role in the proliferation and activation of T cells and natural killer cells.
Automatically generated - may contain errors
Lab products found in correlation
Rhil 7, by Thermo Fisher Scientific (13 mentions)
Rhil 2, by Thermo Fisher Scientific (10 mentions)
Rhil 21, by Thermo Fisher Scientific (5 mentions)
Rhil 12, by Thermo Fisher Scientific (2 mentions)
Rhil 1β, by Thermo Fisher Scientific (2 mentions)
Rhil 23, by Thermo Fisher Scientific (2 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (2 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Rpa t4, by Thermo Fisher Scientific (2 mentions)
Bv711 clone a019d5, by BioLegend (2 mentions)
Fitc conjugated cd127 ebiordr5, by Thermo Fisher Scientific (2 mentions)
Pe cy7 conjugated foxp3 pch101, by Thermo Fisher Scientific (2 mentions)
V450 conjugated active caspase3 c92 605, by BD (2 mentions)
Pe conjugated p y705 stat3 4 p stat3, by BD (2 mentions)
Pe conjugated p y694 stat5 47 stat5, by BD (2 mentions)
Pe conjugated ctla 4 14d3, by Thermo Fisher Scientific (2 mentions)
Rhil 6, by BD (2 mentions)
Rhflt 3l, by Thermo Fisher Scientific (2 mentions)
Rhil 18, by R&D Systems (2 mentions)
33 protocols using rhil 15
1
Peripheral Blood Mononuclear Cell Activation Protocol
2
Decidual NK cell differentiation assay
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MMC were stimulated in vitro with medium as a control, with anti-CD3/anti-CD28 mAb-coated microbeads in a 1:10 bead-to-cell ratio (Invitrogen, Bleiswijk, The Netherlands) alone, rhIL-15 (100 ng/ml; Gibco Life Technologies) alone, and beads together with rhIL-15 in 96-well U-bottom plates. The rationale for using IL-15 is because IL-2 is hardly detected in decidua and after implantation, endometrial NK cells start to differentiate as a result of local IL-15 production9 (link). After 5 days of culture at 37 °C in a humidified 5% CO2 incubator, cells were harvested and the presence of T cell subsets and differentiation was measured with flow cytometry.
3
Antigen-Specific T Cell Generation
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Antigen specific T cells were generated by stimulation of the lymphocyte population with peptide pulsed moDCs at a T cell to antigen presenting cell (APC) ratio of 5:1. Where indicated, starting lymphocyte populations were enriched for CD4+ T cells by immunomagnetic negative selection (EasySep Stem Cell Technologies). Cells were co-cultured in cell growth medium supplemented with either standard T cell growth cytokines of 10 ng/ml rhIL-7 and rhIL-15 (Peprotech) or enhanced cytokine conditions of 20 ng/ml rhIL-1β, 20 ng/ml rhIL-6, 10 ng/ml rhIL-7, 10 ng/ml rhIL-15, 50 ng/ml rhIL-21, 25 ng/ml rhIL-23, and 5 ng/ml rhTGFβ (Peprotech). After 72 h of culture, 30 IU/ml rhIL-2 was added to the culture. Cultures were fed or split every 2–3 days as needed. Cultures were re-stimulated up to 2 times with peptide pulsed moDCs every 10–14 days.
4
Naive CD8+ T Cell Activation and Expansion
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Sorted naive CD8+ T lymphocytes were cultured in the completed RPMI 1640 medium supplemented with 10% fetal bovine serum, 50 units/mL penicillin, 50 μg/mL streptomycin, 1 mM sodium pyruvate, 1×MEM with nonessential amino acids and 50 μM β-mercaptoethanol. The CD8+ naive T cells were activated by 2 μg/mL plate-bound anti-CD3 antibody (R&D system, Minneapolis, MN) and 1 μg/mL soluble anti-CD28 antibody (R&D) and then cultured with recombinant human (rh) IL-2, rhIL-7, rhIL-15, or rhIL-21 (PeproTech, Rocky Hill, NJ) in the indicated concentrations. Medium fed with cytokines was replaced every 3–4 days. Cells were counted every 3–4 days.
5
Flow Cytometry Analysis of Immune Markers
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The following directly conjugated Abs were used in flow cytometry: BV711 or FITC-conjugated CD4 [OKT4 (Biolegend) or RPA-T4 (eBioscience)], BV711- (clone A019D5; Biolegend) or FITC-conjugated CD127 (eBioRDR5; eBioscience), PE- (BD Biosciences) or BV421-conjugated (Biolegend) CD25 (MA251), allophycocyanin-conjugated FOXP3 (236A/E7; eBioscience) or PE-Cy7–conjugated FOXP3 (PCH101; eBioscience), V450-conjugated active CASPASE3 (C92-605; BD Biosciences), PE-conjugated p-(Y705)STAT3 (4/P-STAT3; BD Biosciences), PE-conjugated p-(Y694)STAT5 (47/STAT5; BD Biosciences), PE-conjugated CTLA-4 (14D3; eBioscience). Recombinant human IL (rhIL)-2 (Roche), IL-6, rhIL-7, rhIL-15 (Peprotech), and rhIL-6 (BD Biosciences) were used at the concentrations indicated in the figure legends. Tocilizumab (TOC; anti–IL-6R) and infliximab (anti–TNF-α) were used at a final concentration of 10 μg/ml. Cycloheximide (Sigma) was used at a concentration of 50 μg/ml.
6
Cytokine-Mediated PBMC Activation
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Human donor PBMCs (2×106/ml) were cultured in complete RPMI 1640 medium (GibcoBRL) and 10% FCS for 10 days in the presence of α-GalCer along with rhIL-2 (10 ng/ml) in addition to the following cytokines added individually or in combination: rhIL-7 (10 ng/ml), rhIL-15 (10 ng/ml), rhIL-1β (5 ng/ml), rhIL-23 (10 ng/ml) (all from PeproTech).
7
Modulation of Human CD8+ T Cell Activation
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Purified naive and total human CD8+ T cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% L-glutamine, respectively. Cells were seeded at 5 to 10×104 cells into 96-well U bottom plate and stimulated with anti-CD3/CD28 coated microbeads (Dynabeads® T-Activator CD3/CD28; Thermo Fisher Scientific, Waltham, MA, USA) in the absence or presence of the indicated cytokines; recombinant human (rh) IL-1β (25 ng/ml), rhIL-6 (6.25 to 100 ng/ml), rhIL-21 (0.1 to 100 ng/ml), rhIL-7 (50 ng/ml), IFN-α (50 or 100 ng/ml), rhIL-10 (6.25 to 100 ng/ml; all from R&D systems, Minneapolis, MN, USA), rhIL-12 (50 ng/ml), rhIL-15 (50 or 100 ng/ml), rhIL-2 (100 IU/ml, all from PeproTech, Rocky Hill, NJ, USA). In some experiments, cells were stimulated with anti-CD3/CD28 coated microbeads with chemical inhibitors: STAT1 inhibitor Fludarabin, STAT3 inhibitor 5,15-DPP, or STAT5 inhibitor CAS 285986-31-4 (all from Sigma-aldrich, St. Louis, MO, USA) .
8
Cell Proliferation Assay with CFSE
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Sorted cells were first labelled using the CellTrace™ CFSE Cell Proliferation Kit (Invitrogen). CFSE‐labelled cells were cultured in RPMI 1640 medium with 10% FBS and 1% P/S with or without anti‐CD3 antibodies (1 μg mL‐1; BD Biosciences) or rhIL‐15 (20 ng mL‐1, PeproTech) for 48 h at 37°C in a 5% CO2 atmosphere. After incubation, the cells were harvested and stained with fluorochrome‐conjugated antibodies against surface proteins. After washing, samples were acquired on a BD FACSCanto™ II flow cytometer (BD Biosciences) and the frequency of CFSElow cells was determined using FlowJo software (TreeStar).
9
Cytokine Production by PFCs and PBMCs
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PFCs or PBMCs were incubated at a concentration of 2×106/mL with different doses of rhIL-12 (eBioscience San Diego, CA, USA) or rhIL-15 (Peprotech, Rocky Hill, NJ, USA), and cell-free supernatants were collected at different time points and assessed by ELISA for IFN-γ (BD Bioscience Pharmingen) and IL-22 (R&D System) production according to the manufacturer’s protocols, respectively. For the detection of intracellular cytokines, the cells were cultured for 8 h in the presence of berfeldin A (BFA, 10μg/mL; Sigma-Aldrich, St Louis, MO, USA) and then subjected to flow cytometry analysis. PFCs or PBMCs were incubated at a concentration of 2×106/mL with BCG (20 μg/mL, Chengdu Institute of Biological Products, Chengdu, China) or M. tb-Ag for 8 h in the presence of BFA for the detection of IFN-γ, IL-22 and IL-17 by flow cytometry. Sorted CD45RO+ or CD45RO- NK cells were treated with or without BCG for 48 h. Sorted CD45RO+ or CD45RO- NK cells were co-cultured with purified CD14+ cells (at ratio of 4: 1) in the presence or absence of BCG, BCG plus 2B10 (anti-IL-12Rβ1 mAbs, 10μg/mL, Hoffmann-La Roche Inc., USA), LPS (100ng/mL, Sigma-Aldrich, St Louis, MO, USA) and LPS plus 2B10 for 48 h. Cell-free supernatants were harvested and assessed by ELISA for the production of IFN-γ and IL-22 according to the manufacturer’s protocols, respectively.
10
In Vitro Differentiation of T and NK Cells
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OP9 cells expressing Delta-like 4 (OP9-DL4) were generated and described previously6 , 7 . 5–20 × 104 isolated CD34+CD43− cells were added to individual wells of a 6-well plate containing OP9-DL4 cells, and cultured with: T cells: rhFlt-3L (5 ng/mL) and rhIL-7 (5 ng/mL); NK cells: rhFlt-3L (5 ng/mL) and rhIL-15 (10 ng/mL) (Peprotech, Rocky Hill, NJ). rhSCF (100 ng/mL) was added to both cultures for the first 6 days. Every five days co-cultures were transferred onto fresh OP9-DL4 cells by vigorous pipetting and passaging through a 40μm cell strainer. Cells were analyzed by flow cytometry on the days indicated. Populations scored as positive yielded greater than 100 gated CD45+ events.
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