The human NK cell line NK-92 was maintained in RPMI-1640 medium (Invitrogen), supplemented with 50 mg/ml penicillin, 50 mg/ml streptomycin, 20% heat-inactivated FBS (Invitrogen), and 150 U/mL rhIL-2 (Hoffman-LaRoche). Before cytokine stimulation, NK-92 cells were starved for IL-2 for ~24 h. NK-92 and purified primary NK cells were next incubated in RPMI-1640 media plus 20% FBS at 37°C with or without the addition of rhIL-12 (10 ng/mL) and/or rhIL-18 (100 ng/mL) (R&D Systems) for 24 h or the indicated time.
Rhil 18
Manufactured by R&D Systems
Sourced in United States
RhIL-18 is a recombinant human interleukin-18 (IL-18) protein. IL-18 is a pro-inflammatory cytokine that plays a role in the immune response.
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Lab products found in correlation
Rhil 12, by R&D Systems (4 mentions)
Rhil 12, by Thermo Fisher Scientific (2 mentions)
Rhil 15, by Thermo Fisher Scientific (2 mentions)
Heat inactivated fbs, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Rhil 2, by Roche (1 mentions)
Interleukin 2 (il 2), by R&D Systems (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Intracellular ifn γ, by BD (1 mentions)
Anti human cd107a b abs, by BD (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Brefeldin a, by BD (1 mentions)
Monensin, by Merck Group (1 mentions)
Cd3 pe cy7, by Thermo Fisher Scientific (1 mentions)
Cd56 ecd, by Beckman Coulter (1 mentions)
Tnf α fitc, by BD (1 mentions)
Cd16 apc cy7, by BD (1 mentions)
Live dead stain, by Thermo Fisher Scientific (1 mentions)
Ifnγ v450, by BD (1 mentions)
Live dead stain, by BD (1 mentions)
10 protocols using rhil 18
1
Cytokine stimulation of NK-92 cells
2
Cytokine-Induced NK Cell Activation Assay
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Cultures were performed in RPMI1640-Glutamax supplemented with 1% non-essential amino acids, 100μg/mL gentamycin, 10% fetal calf serum (Invitrogen) and 1mM sodium pyruvate (SigmaAldrich). PBMCs were cultured in the presence or absence of recombinant human interleukin (rhIL)12 and rhIL18 (R&D Systems) for 18h. To measure IFN-γ secretion, 1μl/ml brefeldinA (BD) was added for the last 4h. Cells were labeled with CD3, CD16, and CD56 Abs (BD) and stained for intracellular IFN-γ (BD). NK cytotoxic activity was evaluated by a CD107 degranulation assay. The cultures were washed and co-cultured with K562 cells (5:1 ratio) for 3h. Anti-human CD107a/b Abs (BD) were added at the start of the re-stimulation together with GolgiSTOP for the last 2h. The cells were labeled with CD3, CD16, and CD56 Abs before flow cytometry analysis.
3
Cytokine Production in PBMCs
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For intracellular staining for IFN-γ production, PBMCs were incubated with 50 ng/ml of rhIL-12 (Miltenyi Biotec) and 50 ng/ml of rh-IL18 (R&D Systems) for 21 h at 37°C. One mM monensin (Sigma-Aldrich, Gillingham, UK) was added for the final 3 h. Cells were stained with antibodies to CD3-PE-Cy7 (eBioscience), CD16-APC-Cy7 (BD Biosciences) and CD56-ECD (Beckman Coulter) and subsequently fixed and permeabilized, followed by intracellular staining for IFN-γ-V450 (BD Biosciences). Dead cells were excluded by live/dead stain (Invitrogen).
For TNF-α production, PBMCs were stimulated with phorbol myristate acetate (PMA) (3 ng/ml) and ionomycin (100 ng/ml) (Sigma-Aldrich) for 3 h in the presence of 1 mM of monensin (Sigma-Aldrich). Cells were stained with antibodies to CD3-PE-Cy7 (eBioscience), CD16-APC-Cy7 (BD Biosciences) and CD56-ECD (Beckman Coulter) in the presence of live/dead stain followed by fixing, permeabilization and intracellular staining for TNF-α-FITC (BD Biosciences).
For TNF-α production, PBMCs were stimulated with phorbol myristate acetate (PMA) (3 ng/ml) and ionomycin (100 ng/ml) (Sigma-Aldrich) for 3 h in the presence of 1 mM of monensin (Sigma-Aldrich). Cells were stained with antibodies to CD3-PE-Cy7 (eBioscience), CD16-APC-Cy7 (BD Biosciences) and CD56-ECD (Beckman Coulter) in the presence of live/dead stain followed by fixing, permeabilization and intracellular staining for TNF-α-FITC (BD Biosciences).
4
Phenotypic Characterization of NK Cells
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Peripheral blood mononuclear cells were surface stained with CD3-BV510, CD16-FITC, CD56-V450, NKG2D-PerCP-eFluor710 (eBioscience), and TRAIL-PE (R&D systems) as previously described (44 (link), 45 (link)). To measure the frequency of IFN-γ-producing NK cells and NK cell degranulation, PBMC were incubated for 6 h with rhIL-12 and rhIL-18 (R&D systems) and CFSE-stained K562 (E:T 5:1), respectively. CD107a-APC was added 2 h after the start of the culture to the PBMC: K562 cultures. A protein inhibitor cocktail (eBioscience) was added to all cultures 3 h from the start of the culture. PBMC were then surface stained as described above with CD3, CD16, and CD56 antibodies. The rhIL-12- and rhIL-18-stimulated wells were stained intracellularly with IFN-γ-PE-Cy7 as previously described (44 (link), 45 (link)). PBMC were also stimulated with PMA/ionomycin as a positive control. The gating strategy to assess the ex vivo frequency of cytokine-producing (CD56bright, CD16−) and cytotoxic (CD56dim, CD16+) NK subsets is described in Figure S1 in Supplementary Material.
5
Multiparametric Analysis of Immune Cells
6
Cytotoxicity Assay of HIV Drugs
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rhIL-2 and rhIL-15 were provided by the BRB/NCI Preclinical Repository. rhIL-12 was obtained from PeproTech and rhIL-18 from R&D systems. HODHBt was purchased from AK Scientific. K562-green fluorescent protein (GFP) (ATCC CCL-242-GFP) cell culture was maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), glutamine, and penicillin-streptomycin. A2780 cell culture was maintained in RPMI 1640 medium supplemented with 10% FBS, glutamine, and penicillin-streptomycin. U87 cell culture was maintained in DMEM supplemented with 10% heat-inactivated FBS, glutamine, OCILy1 and OCILy10 cell cultures were maintained in Iscove’s modified Dulbecco’s medium (IMDM) medium supplemented with 10% human serum, glutamine, and penicillin-streptomycin. Nelfinavir and raltegravir were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, and HIV-1NL4-3 from Malcolm Martin.
7
NK Cell-Mediated Inhibition of HIV Infection
8
Stimulation of NK Cells with B. pertussis
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NK cells were seeded into round-bottom 96-wells plates (Greiner) at 150,000 cells/well in IMDM culture medium supplemented with 5 ng/ml recombinant human IL-15 (rhIL-15; PeproTech) (29 (link)). Next, NK cells were immediately stimulated with B. pertussis B4393 at a MOI of 10 in the presence or absence of 5 ng/ml recombinant human IL-18 (rhIL-18; R&D systems), 10 ng/ml rhIL-6 (Miltenyi Biotec), 10 ng/ml rhTNFα (PeproTech), or 10 ng/ml rhIL-1β (InvivoGen). Stimulations were performed at 37°C and 5% CO2 for 18 h after which BD GolgiPlugTM containing Brefeldin A (BD Biosciences) was added to the culture for 4 h, to inhibit cytokine secretion, before collecting the NK cells for flow cytometry analysis.
9
Cytokine-Mediated Whole Blood Stimulation
10
Isolation and Activation of NK Cells
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After PBMC isolation, NK cells were magnetically isolated using an NK isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) via negative selection following the manufacturer’s instructions. NK cells were counted and checked for purity (CD3- CD56+ ≥ 90%). They were resuspended in RPMI medium with 15% FCS, 100 units/mL of penicillin, 0.1 mg/mL of streptomycin, and 2 mM of glutamine and distributed in a 24-well plate (1 × 105 cells/well). NK cells were either left unstimulated or stimulated with one of the following: rhIL-15 (100 ng/mL) (R&D Systems) + rhIL-1β (10 ng/mL) (Peprotech, Cranbury, NJ, USA), rhIL-15 (100 ng/mL) + rhIL-18 (100 ng/mL) (R&D Systems), and rhIL-2 (10 ng/mL) + rhIL-12 (10 ng/mL) (Peprotech). Cells were incubated for 3 days at 37 °C with 5% CO2.
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