DC were generated from monocytes using a standard protocol [27 (link)]. Briefly, monocytes were isolated by plastic adherence and cultured for 5 days in the presence of 800 IU/ml GM-CSF and 250 IU/ml IL-4 (Peprotech). On day 5, non-adherent DC were harvested, loaded with peptide antigens and SEV, and matured overnight. CMV peptides were used at 1 μg/ml, SEV were added at 105 per cell, and the maturation stimulus was monocyte-conditioned media mimic (MCM mimic), consisting of a final concentration of 50 ng/ml TNF-α, 50 ng/ml IL-1β, 1.5 μg/ml IL-6 and 10 μg/ml prostaglandin E2 (all Peprotech). DC phenotype for some experiments was confirmed by assessing cells using flow cytometry (high HLA-DQ, low or absent CD14, moderate CD80). Matured, antigen-loaded DC were washed before use in experiments.
Prostaglandin e2
Manufactured by Thermo Fisher Scientific
Prostaglandin E2 is a chemical compound used in laboratory research. It is a member of the prostaglandin family and plays a role in various physiological processes.
Automatically generated - may contain errors
Lab products found in correlation
Il 1β, by Thermo Fisher Scientific (3 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (3 mentions)
Tnf α, by Thermo Fisher Scientific (2 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Prostaglandin e2, by Merck Group (1 mentions)
Rpmi 1640, by Lonza (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
L glutamine, by Lonza (1 mentions)
Naive cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Recombinant human interleukin 4, by Thermo Fisher Scientific (1 mentions)
3 protocols using prostaglandin e2
1
Generation of Antigen-Loaded Dendritic Cells
2
Generation of Monocyte-Derived Dendritic Cells
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PBMCs were isolated by gradient-density centrifugation. 5 × 107 PBMCs in 10 ml DMEM were adhered to a human IgG-coated tissue culture dish (BD Falcon) and incubated for 1 h at 37°C. Then, the medium was replenished with 10 ml moDC medium [RPMI-1640/containing 1% penicillin/streptomycin, 1% l -glutamine, 1% HEPES, and 2% human sera type AB (Lonza)]. On the next day, the medium was replenished with fresh moDC medium containing 800 U/ml GM-CSF and 50 U/ml IL-4. On day 3 and day 5, 4 ml of fresh moDC medium was added containing 800 U/ml GM-CSF/50 U/ml IL-4 and 400 U/ml GM-CSF/25 U/ml IL-4, respectively. On day 6, half of the cells were matured using a maturation cocktail consisting of 13.2 ng/ml IL-1β, 10,000 U/ml IL-6, 10 ng/ml TNFα, and 1 µg/ml prostaglandin E2 for 24 h. IL-1β, IL-6, and TNFα were purchased from PeproTech and prostaglandin E2 from Sigma Aldrich.
3
Isolation and Maturation of Human Dendritic Cells
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For the isolation and cultivation of DCs, human peripheral blood mononuclear cells (PBMCs) were extracted from healthy participants and seeded. After 2 h, the suspended cells were carefully removed by washing, and recombinant human granulocyte-macrophages (GM-CSF) (1,000 U/ml, PeproTech, 300-03) and recombinant human interleukin 4 (IL-4) (500 U/ml, PeproTech, 200-04) were added to the adherent cells. On the 6th day of induction culture, recombinant human tumour necrosis factor (10 ng/ml, PeproTech, 300-01A), IL-1β (10 ng/ml, PeproTech, 200-01B), IL-6 (1000 U/ml, PeproTech, 200-06) and prostaglandin E2 (1 µg/ml, PeproTech, 3632464) were used to induce the maturation of DCs. On days 7–8 of induction culture, mature DCs were harvested. Flow cytometry was performed to detect the expression of CD80 (FITC conjugate; BioLegend, 305205, RRID: AB_314501), CD83 (PE conjugate; BioLegend, 305307, RRID: AB_314515) and CD86 (APC conjugate; BioLegend, 374207, RRID: AB_2721448).
Human PBMCs were separated, and a Naive CD4+ T Cell Isolation Kit (Miltenyi Biotec, 130-091-894) was used to sort and culture the initial T cells.
The DCs described above were cocultured with initial T cells at a 1:1 ratio for 48 h. The proportion of Tregs was analysed using flow cytometry.
Human PBMCs were separated, and a Naive CD4+ T Cell Isolation Kit (Miltenyi Biotec, 130-091-894) was used to sort and culture the initial T cells.
The DCs described above were cocultured with initial T cells at a 1:1 ratio for 48 h. The proportion of Tregs was analysed using flow cytometry.
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