Generation of Tolerogenic Monocyte-Derived Dendritic Cells

Monocytes from LRSC were isolated by gradient centrifugation with Ficoll-Paque Plus (GE Healthcare) and, subsequently, Percoll gradient separation (GE Healthcare). After 2 h at 37°C, non-adherent cells were removed, and complete medium containing 1,000 U/ml GM-CSF and 800 U/ml IL-4 (Miltenyi) was added. One micromole of Dexamethasone (Dex) (Sigma) was added at day 3 or day 6 of differentiation to generate tolerogenic DC as described by Cabezón et al. (5 (link)). moDC phenotype at the end of differentiation was validated by flow cytometry monitoring of CD1a, CD14, and CD11c markers (Supplementary Figure 1).

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Giroud P., Renaudineau S., Gudefin L., Calcei A., Menguy T., Rozan C., Mizrahi J., Caux C., Duong V, & Valladeau-Guilemond J. (2020). Expression of TAM-R in Human Immune Cells and Unique Regulatory Function of MerTK in IL-10 Production by Tolerogenic DC. Frontiers in Immunology, 11, 564133.

Publication 2020

Ficoll-Paque Plus Percoll gradient separation GM-CSF IL-4 Dex

Cd1a Cells Centrifugation Dexamethasone Ficoll Flow cytometry Gm csf Monocytes Percoll Phenotype Tolerogenic

Corresponding Organization : Cherry Biotech (France)

Other organizations : Université Claude Bernard Lyon 1, Centre Léon Bérard, Inserm, Centre de Recherche en Cancérologie de Lyon, Centre National de la Recherche Scientifique

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Variable analysis

independent variables

Timing of dexamethasone (Dex) addition: day 3 or day 6 of differentiation

dependent variables

Monocyte-derived dendritic cell (moDC) phenotype at the end of differentiation, as validated by flow cytometry monitoring of CD1a, CD14, and CD11c markers

control variables

Monocytes isolated by gradient centrifugation with Ficoll-Paque Plus and Percoll gradient separation
Culture conditions: 2 h at 37°C, complete medium containing 1,000 U/ml GM-CSF and 800 U/ml IL-4

controls

No positive or negative controls explicitly mentioned

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