Journal: Frontiers in Cellular Neuroscience

Article Title: Mapping the Plasticity of Morphology, Molecular Properties and Function in Mouse Primary Microglia

doi: 10.3389/fncel.2021.811061

Figure Lengend Snippet: Neurotoxicity of microglial phenotypes induced by IFN-γ, IL-4, or IL-10. (A) Experimental scheme to monitor neurotoxicity during activation. Microglia underwent stimulation with IFN-γ, IL-4, or IL-10 for 2–48 h, then the medium was changed and the cells were cultured with fresh medium for 24 h. The microglia-conditioned medium (M-CM) was collected and configured as a proliferation medium for neural stem/precursor cells (NSPCs). The NSPCs were cultured with M-CM for 24 h and the apoptotic NSPCs were detected. (B) Representative micrographs showing immunocytochemical labeling for cleaved caspase-3 in NSPCs treated for 24 h with conditioned medium from IFN-γ-, IL- 4-, or IL-10-stimulated microglia. (C) Quantification of the percentages of cleaved caspase-3 + -DAPI + cells following treatment with conditioned medium from IFN-γ-, IL-4- or IL-10-stimulated microglia for 2–72 h. Five, 40× micrographs were taken from each well, and all cells in the field were measured. Data are presented as mean ± SEM ( n = 4–6 well per group), ** P < 0.01, *** P < 0.005 (one-way ANOVA with Tukey’s multiple-comparisons test).

Article Snippet: Microglia were plated at a density of 5 × 10 5 cells/cm 2 and treated with each of the cytokines as described in Section “Stimulation of Primary Microglia,” followed by washing with PBS twice, and then addition of DMEM-F12 + GlutaMax medium for another 24 h. The microglial medium was collected and used as conditioned medium to culture NSPCs for 24 h. Apoptosis of NSPCs were labeled overnight using the mouse anti-cleaved caspase-3 antibody (1:300; Cell Signaling Technology, United States), followed by the anti-mouse secondary antibodies mentioned in Section “Immunofluorescence.” Finally, cells were stained for 5 min with DAPI (1:10,000) and imaged using a fluorescence microscope (Olympus BX51, Japan).

Techniques: Activation Assay, Cell Culture, Labeling

Journal: Frontiers in Cellular Neuroscience

Article Title: Mapping the Plasticity of Morphology, Molecular Properties and Function in Mouse Primary Microglia

doi: 10.3389/fncel.2021.811061

Figure Lengend Snippet: Changes in neurotoxicity of IFN-γ, IL- 4-, or IL-10-treated microglia in different response state. (A–C) Changes in neurotoxicity of IFN-γ-treated microglia on NSPCs in different response state following IL-4 or IL-10 exposure for 12 h. (A) Experimental scheme to monitor neurotoxicity during activation. Microglia underwent stimulation with IFN-γ, IL-4, or IL-10 for 2–48 h, then the medium was changed and the cells were stimulated with another cytokine for 12 h. After then the cells were cultured with fresh medium for 24 h. The microglia-conditioned medium (M-CM) was collected and configured as a proliferation medium for neural stem/precursor cells (NSPCs). The NSPCs were cultured with M-CM for 24 h and the apoptotic NSPCs were detected. (B) Representative micrographs showing immunocytochemical labeling for cleaved caspase-3 in NSPCs treated for 24 h with conditioned medium from the reprogrammed microglia. (C) Quantification of the percentages of cleaved caspase-3 + -DAPI + cells following treatment with conditioned medium from the reprogrammed microglia. (D–F) Changes in neurotoxicity of IL-4-treated microglia on NSPCs in different response state following IFN-γ or IL-10 exposure for 12 h. (G–I) Changes in neurotoxicity of IL-10-treated microglia on NSPCs in different response state following IFN-γ or IL-10 exposure for 12 h. Five 40× photographs were taken from each well, and all the cells in the field were measured. Data are presented as the mean ± SEM. n = 4–5 well per group, * P < 0.05, ** P < 0.01, *** P < 0.005 (one-way ANOVA with Tukey’s multiple-comparisons test).

Article Snippet: Microglia were plated at a density of 5 × 10 5 cells/cm 2 and treated with each of the cytokines as described in Section “Stimulation of Primary Microglia,” followed by washing with PBS twice, and then addition of DMEM-F12 + GlutaMax medium for another 24 h. The microglial medium was collected and used as conditioned medium to culture NSPCs for 24 h. Apoptosis of NSPCs were labeled overnight using the mouse anti-cleaved caspase-3 antibody (1:300; Cell Signaling Technology, United States), followed by the anti-mouse secondary antibodies mentioned in Section “Immunofluorescence.” Finally, cells were stained for 5 min with DAPI (1:10,000) and imaged using a fluorescence microscope (Olympus BX51, Japan).

Techniques: Activation Assay, Cell Culture, Labeling