Mature and immature monocyte-derived dendritic cells (mDC and iDC, respectively) were generated from magnetically isolated CD14+ monocytes (26 (link)). The cells were cultured in complete medium (RPMI 1640 with 10% FCS, 2 mM L-glutamine and 100 IU/ml penicillin, 100 μg/ml streptomycin) supplemented with IL-4 and GM-CSF (both at 50 ng/ml; Immunotools) for 7 days. MSC-EVs at a concentration of 20 μg/ml were added to the DC generation culture on day 3, when fresh medium containing the same concentrations of IL-4 and GM-CSF was replenished. Mature DCs were generated by adding LPS (100 ng/ml; Sigma) to the DC culture on day 6. The EV-treated immature and mature DCs (iDC-EV and mDC-EV) were harvested on day 7 and used in the subsequent experiments.
Il 4 and gm csf
Manufactured by Immunotools
Sourced in Germany
IL-4 and GM-CSF are two cytokines that play important roles in the immune system. IL-4 is a key regulator of B-cell and T-cell differentiation, while GM-CSF is involved in the regulation of granulocyte and macrophage production. These cytokines are commonly used in laboratory research to study their effects on various immune cell types.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Rpmi 1640, by Immunotools (1 mentions)
Rpmi 1640 glutamax, by Thermo Fisher Scientific (1 mentions)
Penicillin g streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Gmp dc medium, by CellGenix (1 mentions)
Lymphoprep, by Abbott (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
24 well culture plate, by Corning (1 mentions)
Macs ls column, by Miltenyi Biotec (1 mentions)
4 protocols using il 4 and gm csf
1
Generation and Characterization of Monocyte-Derived Dendritic Cells
2
Dendritic Cell Preparation from Bone Marrow
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For bone marrow–derived dendritic cell (BMDC) preparation, cells were isolated and cultured according to a standard method with minor modifications (Han et al., 1999 (link)). Briefly, bone marrow cells were cultured in RPMI 1640 media supplemented with 1% penicillin and streptomycin, 10% of fetal calf serum (S0115, EMD Millipore, United States), 20 ng/ml of mouse recombinant IL-4 and GM-CSF (ImmunoTools, Friesoythe, Germany). At day 8, non-adherent cells were transferred to a fresh plate, primed by 500 μg/ml lipopolysaccharide (LPS, Sigma) for another 24 h, and used for MLR. In BMDC stimulation assays, non-adherent cells were transferred to 12-well plate at 2 × 106/ml at day 8, stimulated with indicated stimuli for 24 h, and analyzed by flow cytometry.
3
Monocyte-derived Dendritic Cell Culture
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Monocytes were isolated from buffy coats of healthy blood donors using RosetteSep™ human monocyte enrichment antibody cocktail (STEMCELL Technologies), as previously described53 . DC cultures were set up with 60–100 × 106 human peripheral blood monocytes, plated at 20 × 106 cells per 100 mm diameter petri dishes, in DC differentiating media - RPMI-1640 + Glutamax (Invitrogen) cell culture media, with 10% heat inactivated FBS (Lonza) and 1% penicillin G-streptomycin (P/S; Invitrogen), further supplemented with 50 ng/mL IL-4 and GM-CSF (Immunotools). After 4 days of differentiation, cells were washed with PBS and re-cultured for 3 additional days in EV producing media. This media was obtained from ultracentrifuged (100,000 × g, for 2 h at 4 °C) RPMI supplemented with 10% FBS, which was then diluted to 1% FBS, and further supplemented with 1% P/S, IL-4 and GM-CSF, as above. Absence of bovine-EV in the ultracentrifuged RPMI + 10% FBS media was confirmed by transmission electron microscopy (Supplementary Fig. S3 ). Cells were maintained in a humidified incubator, at 37 °C and 5% CO2. DC cultures in EV-producing media were monitored by optical microscopy and no changes in cell morphology or general culture condition were observed (Supplementary Fig. S4 ).
4
Generation of Tolerogenic Dendritic Cells
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Peripheral blood mononuclear cells (PBMC) were isolated by gradient density centrifugation using Lymphoprep (Alere). Monocytes were further separated from PBMC by positive selection using CD14+ magnetic microbeads and a MACS LS column (Miltenyi) according to manufacturer’s instructions. Monocytes were seeded into 24-well culture plates (Corning) at 0.5 x 106/ml (total of 1 ml per well) in GMP DC medium (CellGenix) containing IL-4 and GM-CSF (both at 50 ng/ml; Immunotools) and cultured for 7 days at 37°C, 5% CO2. On day 3, IL-4 and GM-CSF were refreshed at 50 ng/ml final concentration, and half of the medium was replaced with fresh GMP DC medium. To generate tolDC, dexamethasone (Sigma) was added at 10-8 M on days 3 and 6 of culture, and 10-10 M 1,25-dihydroxyvitamin D3 (either Calcitriol from Tocris or Decostriol from Mibo) and 1 µg/ml of the TLR4 agonist monophosphoryl A (MPLA; Avanti) on day 6. To generate mature DC (matDC), cells were treated with 1 µg/ml MPLA (Avanti) on day 6 of culture only. In addition, on day 6 tolDC were labelled with 19F-NP (1.1, 2.2 or 4.4 mg/ml) or left unloaded as a control. DC were harvested on day 7, resuspended in HBSS + 1% FCS in a 30 ml universal tube (Starlab) and placed on ice for further processing (see sections below). Viability and cell numbers were determined by trypan blue (Sigma).
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