Different LM strains: the pathogenic LMWT as well as the mutant strains LMDLLO, LMDActA, and LMDplcADplcB. Our phagocytic functional research integrated evaluation of cell surface markers, transcriptional responses with differential displayed microarrays, cytokine measurements, microbicidal mechanisms, and protein composition of isolated phagosomes or postnuclear supernatants (PNS).Components and MethodsAnimalsWe made use of C57BL/6 mice from our animal facilities at the University of Cantabria. Bone marrow-derived macrophages (BMDMs) were obtained from femurs of 8- to 12-week-old female mice, cultured, and differentiated in Dulbecco’s Modified Eagle’s Medium supplemented with 20 fetal calf serum, 1 mM glutamine, 1 mM NEAA, 25 ng/mL macrophage colony-stimulating factor, 50 mg/mL gentamicin, and 30 mg/mL vancomycin for 7 days. Newborn C57BL/6 pups were utilised to obtain cerebellum for preparation of mixed microglia and subsequent isolation of principal microglial cultures.Mixed Microglial Cell Cultures, Purified Primary Microglia, and Cell LinesMicroglial cultures have already been described previously (Lopez-Fanarraga et al., 2007; Ribes et al., 2010; Scheffel et al., 2012). Detailed procedures for getting mixed microglial cell cultures, purified principal microglia, the microglial cell line BV2, and the murine macrophage cell line J774 are described in Supporting Info.actA BMDM CCL CFU CNS FITC HBE hly H2O2 IFN IL i.p. LLO LM LPS MCP MHC-II NEEA NO NOD PI3K PE PNS TLR TRITCLM gene coding for ActA, a LM protein accountable for actin polymerization bone-marrow derived macrophage chemokine CC ligand colony forming units central nervous program fluorescein isothiocyanate HEPES buffer with EDTA LM gene coding for listeriolysin O hydrogen peroxide interferon interleukin intra-peritoneal listeriolysin O protein Listeria monocytogenes lipopolysaccharide monocyte chemotactic protein significant histocompatibility class II molecules nonessential amino acids nitric oxide nucleotide-binding and oligomerization domain receptor phosphatidylinositol 3 kinase phycoerithrin postnuclear supernatants Toll-like receptor tetramethylrodamine B isothiocyanateBacteriaProf.3-Aminobutan-2-ol In stock D.Tri(1-adamantyl)phosphine site A. Portnoy (University of California. Berkeley, CA) supplied L. monocytogenes 10403S pathogenic strain (LMWT) plus the mutant strains LMDLLO, LMDActA, and LMDplcADplcB. GFP-LM DH-L1039 (GFP-LM) derived from the 10403S LM strain was a gift from Prof.PMID:33712482 D.E. Higgins (Harvard Healthcare College, Boston, MA).Kinetic Infection AssaysCell lines, mixed microglial cultures, BMDMs, and purified primary microglia have been infected as described in Supporting Information and previously (Carrasco-Marin et al., 2009, 2011). Replication indexes (RIs) have been calculated as previously reported (Alvarez-Dominguez and Stahl, 1999; Alvarez-Dominguez et al., 2000; Carrasco-Marin et al., 2009, 2012; Del Cerro-Vadillo et al., 2006; Prada-Delgado et al., 2001).Phagosome and Endosome Isolation and Protein Composition AnalysisBV2 and J-774 cells had been infected with LM strains (LMWT, LMDLLO, LMDActA, or LMDplcADplcB mutants) for 20 min (ten:1;Volume 62, No.Frande-Cabanes et al.: Microglia, the Innate Immune Cellsbacteria:cell ratio) and phagosomal fractions have been isolated from thawed PNSs as previously described (Alvarez-Dominguez et al., 1999; Carrasco-Marin et al., 2009, 2011, 2012; Del Cerro-Vadillo et al., 2006; Prada-Delgado et al., 2001). Endosomes type noninfected BV2 and J-774 cells have been obtained as described (Del CerroVadillo et al., 2006; Prada-Delgado.