Cd11c clone 3

In all, 8*10⁵ single cells from fresh human brain material and cerebral organoids were stained in a 96-well V-bottom plate using the following antibodies from Ebioscience (San Diego, CA): CD45 (#119459), CD11b (#12011841), CD14 (clone 61D3, #90170149025), CD11c (clone 3.9, #11011641), HLA-DR (clone LN3, #17995641), CX3CR1 (clone eB149/10H5, #17609941), IgG1 (#114714 and #17471441), IgG2b (#17403181); antibodies from BD Bioscience: CD206 (#561763); antibodies from BioLegend IgG2b (#400319). Protein expression was quantified on a FACSCanto II (BD Biosciences) and analyzed with FACSDiva software (Version 8.0.1, BD Biosciences). Protein expression was measured in cells that were gated to be alive, single, and CD11b⁺ (Supplementary Fig. 5b). The geoMFI mean was subtracted from the geoMFI of the corresponding isotype control.

Monocyte derived DC were generated by standard methods [38 (link)] from PBMC obtained from anonymous donors (Continental Blood Services Inc, Miami) and plated on 6 well plates. DC were then transduced with Ad5-ΔLMP1-MAVS or Ad5-Gag control (MOI = 50). DC were incubated with virus at 4°C for 1 h, followed by 3 h at 37°C. Complete media was then added to 2ml. As a positive control, DC were matured with cytokine mix Mimic (5 ng/ml TNF-α (Peprotech), 5ng/ml IL-1b (Peprotech), 750ng/ml IL-6 (Peprotech), and 1ug/ml PGE2 (Sigma)). Cells were incubated for 36 hours at 37°C, harvested, and stained with the following antibodies: CD86 clone 2331 (FUN-1), CD80 clone L307.4, HLA-DR clone TU36, CD83 clone HB15e, CD40 clone 5C3, CD197 (CCR7) clone 3D12, and CD11c clone 3.9) (BD Bioscience). After flow cytometry analysis, the mean fluorescence intensity (MFI) for each antibody was calculated for CD11c+ dendritic cells under each experimental condition. FlowJo 7.6.4 flow cytometry analysis software (FlowJo, Ashland, OR) was used for analysis. Three independent wells were analyzed for each condition.

MDDC were matured and then stained for markers of maturation and activation (anti-human CD14 clone M5E2, CD86 clone 2331 (FUN-1), CD80 clone L307.4, HLA-DR clone TU36, CD83 clone HB15e, CD40 clone 5C3, CD197 clone 3D12, and CD11c clone 3.9) (BD Bioscience).