Additional file 4: of CRAMP deficiency leads to a pro-inflammatory phenotype and impaired phagocytosis after exposure to bacterial meningitis pathogens

Exogenous application of CRAMP reduced NFκB translocation in CRAMP-KO microglial cells. Microglial cells from CRAMP-WT (A) or KO (B) mice were incubated with 1, 2 or 10 μM mouse CRAMP with or without supernatant of NM for 30 min, 1 or 2 h. After incubation cells were fixed and immunolabeled using anti-NFκB p65 antibody (red) and nuclear counterstaining DAPI (blue) and examined with fluorescence microscopy. The figure shows representative results from three independent experiments. Scale bar = 20 μm. (TIFF 19383 kb

Additional file 5: of CRAMP deficiency leads to a pro-inflammatory phenotype and impaired phagocytosis after exposure to bacterial meningitis pathogens

Co-stimulation of exogenous CRAMP and bacterial supernatant NM induced increase of HO-1 immunofluorescence in CRAMP-KO microglial cells. Microglial cells from CRAMP-WT or KO mice were incubated with 1, 2 or 10 μM mouse CRAMP with or without supernatant of NM for 6 h. After incubation cells were fixed and immunolabeled using anti-HO-1 antibody (green) and nuclear counterstaining DAPI (blue) and examined with fluorescence microscopy. The figure shows representative results from three independent experiments. Scale bar = 20 μm. (TIFF 3834 kb

Additional file 3: of CRAMP deficiency leads to a pro-inflammatory phenotype and impaired phagocytosis after exposure to bacterial meningitis pathogens

Proliferation and apoptosis induction after bacterial stimulation in CRAMP-WT or CRAMP-KO microglial cells. Microglial cells from CRAMP-knockout (KO) or wild-type (WT) mice were incubated with bacterial supernatants of Gram-positive bacterium Streptococcus pneumoniae (SP) or Gram-negative bacterium Neisseria meningitidis (NM) and bacterial cell wall components lipopolysaccharide (LPS) or peptidoglycan (PGN) for 24 h. After incubation, glial cells were fixed and immunolabeled using the proliferation marker Ki67 (red), TUNEL reaction mixture for apoptosis and DAPI for nuclear counterstaining (blue). (A) Representative results from one of three independent experiments. (B) Ki67 proliferation index was calculated by the number of positive cells expressing Ki67 divided by the total number of cells in each field. These results were calculated for at least 20 separate cells. Scale bar = 20 μm. (TIFF 6059 kb

Additional file 5: of CRAMP deficiency leads to a pro-inflammatory phenotype and impaired phagocytosis after exposure to bacterial meningitis pathogens

Co-stimulation of exogenous CRAMP and bacterial supernatant NM induced increase of HO-1 immunofluorescence in CRAMP-KO microglial cells. Microglial cells from CRAMP-WT or KO mice were incubated with 1, 2 or 10 μM mouse CRAMP with or without supernatant of NM for 6 h. After incubation cells were fixed and immunolabeled using anti-HO-1 antibody (green) and nuclear counterstaining DAPI (blue) and examined with fluorescence microscopy. The figure shows representative results from three independent experiments. Scale bar = 20 μm. (TIFF 3834 kb

Additional file 1: of CRAMP deficiency leads to a pro-inflammatory phenotype and impaired phagocytosis after exposure to bacterial meningitis pathogens

Additional Material and Methods. Fluorescence microscopy of TUNEL and Ki67: Primary mice astrocytes or microglia were seeded on cover glasses. After stimulation for 24 h, the cells werr fixed with 4% paraformaldehyde. Subsequently, the cells were permeabilized with 0.1% Triton X in 0.1% sodium citrate for 3 min at 4°C. Then, the slices were incubated at 37°C for 1 h with TUNEL reaction mixture according the manufacturer’s protocol (In Situ Cell Death Detection Kit, Roche Diagnostics, Mannheim, Germany). After washing with PBS and blocking with 1.5 BSA in PBS for 10 min, the slices were incubated at 4°C about the night with Ki67 antibody (rabbit polyclonal; ab15580, abcam, UK). Finally, the slices were incubated with anti-rabbit Cy3 (AP132C, Millipore, Darmstadt, Germany) for 1 h at room temperature. Nuclear counter-staining was performed with Diamidino-2-phenylindole dihydrochloride DAPI (Sigma 9542, Munich, Germany). Cells were digitally photographed using a Keyence digital microscope (BZ-9000, Neu Isenburg, Germany). Ki67+ positive cells were counted for each treatment, where five 63×fields were evaluated. The proliferation index was determined by the number of positive cells expressing Ki67 divided by the total number of cells in each field. (DOCX 16 kb