Computational protocol: Macrophage-Derived Extracellular Succinate Licenses Neural Stem Cells to Suppress Chronic Neuroinflammation

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[…] Ex vivo samples were collected at 10 and 30 dpt. Mice were deeply anesthetized with isoflurane (4% induction) and decapitated. The entire brain and spinal cord were exposed, isolated and stored in RNAlater (QIAGEN) at 4°C until use. Samples were homogenated using a potter and total RNA was extracted using the RNeasy Plus Universal Midi Kit (QIAGEN) following manufacturer’s instructions.Total RNA from Mφ or BV2 microglial cell line in co-cultures was collected at given time points. Before collection, cells were washed with PBS, 350 μL of RLT buffer were added, and samples stored at −80°C until extraction.Total RNA after succinate stimulation was collected as it follows. Cells were dissociated, counted, and 1.5x106 cells/ml/well per condition were seeded in a 6 well plate. After 6 hr, sodium succinate dibasic hexa-hydrate (500 μM, Sigma-Aldrich) in PBS or PBS alone (control) was added to each well. After 15 min cells were collected and spun at 16,000 g for 30 s. Pellets were washed with PBS, resuspended in 350 μL of RLT buffer and stored at −80°C until extraction.Total RNA from all in vitro samples was extracted using the RNeasy Micro Kit (QIAGEN) following manufacturer’s instructions.For microarrays, samples were prepared according to Affymetrix protocols (Affymetrix, Santa Clara, CA). RNA quality and quantity were ensured using the Bioanalyzer (Agilent, Santa Clara, CA) and NanoDrop (Thermo Scientific, Waltham, MA) respectively. For RNA labeling, 200 ng of total RNA was used in conjunction with the Affymetrix recommended protocol for the Clariom_S chips. The hybridization cocktail containing the fragmented and labeled cDNAs was hybridized to the Affymetrix Mouse Clariom_S GeneChip. The chips were washed and stained by the Affymetrix Fluidics Station using the standard format and protocols as described by Affymetrix. The probe arrays were stained with streptavidin phycoerythrin solution (Molecular Probes, Carlsbad, CA) and enhanced by using an antibody solution containing 0.5 mg/mL of biotinylated anti-streptavidin (Vector Laboratories, Burlingame, CA). An Affymetrix Gene Chip Scanner 3000 was used to scan the probe arrays. Gene expression intensities were calculated using Affymetrix AGCC software. Downstream analysis was conducted in R/Bioconductor.The annotation package for the Clariom_S chips was generated with pdInfoBuilder (https://www.bioconductor.org/packages/release/bioc/html/pdInfoBuilder.html) using the platform files provided by Affymetrix. The CEL files were then loaded into R, RMA normalized with the oligo package, and filtered to only retain probes annotated as “main” (). Differential expression testing was performed using limma () and the resulting p.values were corrected with the Benjamini-Hochberg method.GO enrichment analyses were performed using the topGO package (https://bioconductor.org/packages/release/bioc/html/topGO.html) with the classic algorithm and Fisher statistic. Microarray heatmaps were generated with the heatmap.2 function of the gplots package with the default clustering methods. The microarray raw data were deposited in ArrayExpress with the accession numbers E-MTAB-5579 and E-MTAB-5586.For qRT-PCR analysis, equal amounts of RNA were reversed-transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions. cDNA was then quantified with the NanoDrop 2000c instrument (Thermo Scientific) and qRT-PCR was performed with the TaqMan® Universal PCR Master Mix (Applied Biosystems) and TaqMan® Gene Expression Assays for: Il12b (Mm01288989_m1, Life Technologies), Il15 (Mm00434210_m1, Life Technologies), Il15ra (Mm04336046_m1, Life Technologies), Cd69 (Mm01183378_m1, Life Technologies), Nos2 (Mm00440502_m1, Life Technologies), Tnf (Mm00443258_m1, Life Technologies), Il1b (Mm00434228_m1, Life Technologies), Bst1 (Mm00477672_m1, Life Technologies), Ust (Mm00616790_m1, Life Technologies) Arg1 (Mm00475988_m1, Life Technologies), Mrc1 (Mm00485148_m1, Life Technologies), Sucnr1 (Mm00519024_m1, Life Technologies), SUCNR1 (Hs00908230_m1, Life Technologies), Ptgs2 (Mm00478374_m1, Life Technologies), and Actb/18S (Life Technologies) were used as internal calibrators. All samples were tested in triplicate on a 7500 Fast Real-Time PCR System (Applied Biosystems) and analyzed with the 2-ΔΔCT method. […]

Pipeline specifications

Software tools Oligo package, limma, TopGO, gplots
Databases ArrayExpress
Application Gene expression microarray analysis
Diseases Phagocyte Bactericidal Dysfunction
Chemicals Dinoprostone, Succinic Acid