Purified NK cells were cultured at 1x106 cells/ml in 200μl RPMI 1640 (Gibco) supplemented with 10% FCS and 1 U/mL Penicillin-Streptomycin (10 000 U/mL, Gibco) in 96 well round-bottom plates. Cells were either left unstimulated or activated with paraformaldehyde inactivated bacteria at MOI 20 (S. pneumoniae, L. monocytogenes or S. agalactiae) and/or with various cytokine cocktails composed of IL-15 (2 ng/ml), IL-18 (1,5 ng/ml) and IL-12 (1,25 ng/ml) (Miltenyi Biotec). Cells were also stimulated with the TLR1/TLR2 agonist Pam3CSK4 (InvivoGen) or with LPS from E. coli O111:B4. After 20 hours of culture at 37°C, 1X Brefeldin-A (BFA solution 1000X, ref no. 420601, BioLegend) was added into each well to block secretion of IFNγ for 4 hours. After incubation at 37°C, cells were collected for intracellular cytokine and cytotoxic proteins by flow cytometry.
Il 18
Manufactured by Miltenyi Biotec
Sourced in Germany
IL-18 is a recombinant human interleukin-18 protein. Interleukin-18 is a proinflammatory cytokine that has a wide range of biological activities.
Automatically generated - may contain errors
Lab products found in correlation
Il 15, by Miltenyi Biotec (3 mentions)
Il 12, by Miltenyi Biotec (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Pam3csk4, by InvivoGen (1 mentions)
Recombinant human il 7, by Miltenyi Biotec (1 mentions)
Mertk, by Cell Signaling Technology (1 mentions)
P mertk, by Abcam (1 mentions)
Granzyme b, by Cell Signaling Technology (1 mentions)
Il 21, by Miltenyi Biotec (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
Mopc 21, by BioLegend (1 mentions)
Cd3 cd28 t activator beads, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Rnalater, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
4 protocols using il 18
1
Activation of NK cells by Bacteria and Cytokines
2
Immunoblotting and Flow Cytometry Antibody Panel
3
PBMC Isolation and T-cell Activation
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Human peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood from consenting healthy donors (protocol approved by the NRES Committee West Midlands ethical board; REC reference 14/WM/1254). Briefly, blood was layered over lymphoprep (Stem Cell Technologies) with resulting PBMC used for subsequent experiments. Plasma was also harvested for CMV IgG (ELISA kit) measurement. For repertoire analysis, all T-cell populations were sorted directly into RNAlater (Sigma) or RLT buffer (Qiagen) supplemented with β-mercaptoethanol (Sigma) on a MoFlo Astrios Cell Sorter (Beckman Coulter). For activation and proliferation of T cells, either PBMC or isolated CD3+ T cells, obtained by positive magnetic bead isolation (Miltenyi), were labelled or not with 0.3 μM CFSE (eBioscience) and cultured with cytokines, 25 ng ml−1 IL-7 (Peprotech), 100 IU ml−1 IL-2, 25 ng ml−1 IL-15, 5 ng ml−1 IL-12, 5 ng ml−1 IL-18 (all Miltenyi), and/or antibodies directed against CD3 (OKT3; eBioscience), CD28 (28.2), mIgG1κ (MOPC-21), TCR γδ (B1; all Biolegend) or CD3/CD28 T activator beads (Invitrogen), where indicated and for up to 7 days in RPMI-1640 medium (Invitrogen) supplemented with 2 mM L -glutamine, 1% sodium pyruvate, 50 μg ml−1 penicillin/streptomycin (Invitrogen) and 10% fetal calf serum (Sigma).
4
Cytokine and Cytolytic Granule Detection Protocol
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To detect cytokines, cells were stimulated overnight with IL-12 (10 ng/mL), IL-15 (50 ng/mL), IL-18 (100 ng/mL) or IL-1β (50 ng/mL), IL-7 (50 ng/mL), IL-23 (50 ng/mL) (Miltenyi Biotec, Bergisch Gladbach, Germany) in the presence of monensin (GolgiStop) or brefeldin (GolgiPlug) (BD Biosciences), respectively.
For intra-cytoplasmic cytokine and cytolytic granules analyses, cells were stained for surface markers and then fixed and permeabilized with Fixation and Permeabilization Kit (BD Biosciences). Then, cells were incubated with cytokine- or Perforin-specific mAbs. To detect TF expression, cells were suspended in 5% BSA buffer, stained for surface markers, subsequently fixed with Transcription Factor Staining Buffer Set and stained for Eomes, AhR and RORγt mAbs.
CD107a degranulation assay-CD56+ obtained lymphocytes in vitro after 27 days of culture were incubated in 1/1 ratio with the melanoma cell line MFO1 or human leukemic cell line K562 in the presence of CD107a mAb for 3-h. monensin (GolgiStop) was added after one hour of incubation to the cells. At the end of the incubation, the cells were collected and marked to surface immunofluorescence and analyzed on the flow cytometer. In each experiment we performed a comparison between CTR and UNC- or GSK-treated cells and then normalized data to compare all experiment together.
For intra-cytoplasmic cytokine and cytolytic granules analyses, cells were stained for surface markers and then fixed and permeabilized with Fixation and Permeabilization Kit (BD Biosciences). Then, cells were incubated with cytokine- or Perforin-specific mAbs. To detect TF expression, cells were suspended in 5% BSA buffer, stained for surface markers, subsequently fixed with Transcription Factor Staining Buffer Set and stained for Eomes, AhR and RORγt mAbs.
CD107a degranulation assay-CD56+ obtained lymphocytes in vitro after 27 days of culture were incubated in 1/1 ratio with the melanoma cell line MFO1 or human leukemic cell line K562 in the presence of CD107a mAb for 3-h. monensin (GolgiStop) was added after one hour of incubation to the cells. At the end of the incubation, the cells were collected and marked to surface immunofluorescence and analyzed on the flow cytometer. In each experiment we performed a comparison between CTR and UNC- or GSK-treated cells and then normalized data to compare all experiment together.
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