Computational protocol: Temporal Changes in Cell Marker Expression and Cellular Infiltration in a Controlled Cortical Impact Model in Adult Male C57BL/6 Mice

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Protocol publication

[…] For cell surface staining, anti-mouse CD45-PE/Cy7, anti-mouse CD11b-PE, anti-mouse granulocyte-differentiation antigen-1 (Gr-1)-FITC, anti-mouse CD11c-Pacific Blue™, and anti-mouse CD3-APC (BioLegend) were used. Anti-mouse CD45-PE/Cy7, anti-mouse CD11b-PE, anti-mouse CD86-APC, and anti-mouse CD206-FITC were also used for detection of M1-like and M2-like microglia. The cells were suspended in flow cytometry staining buffer (BD Biosciences) and treated with Fc-receptor blocker (anti-mouse cluster of differentiation 16/32 (CD16/32) antibody (BioLegend) for 20 min at 4°C in order to eliminate nonspecific binding of antibodies to Fc receptors. Next, the cells were stained by these antibodies for 30 min at 4°C at dark. The samples were washed twice in 2 mL of flow cytometry staining buffer and resuspended in 500 μL of the staining buffer per sample.Flow cytometry was performed with a BD FACSCanto II (BD Bioscience) and analyzed using BD FACSDiva software (BD Bioscience) and the FlowJo software (version 9.3.1; TreeStar, Inc., Ashland, OR). The specificity of the signals of antibodies against specific antigens was determined by performing control experiments using isotype-matched immunoglobulins (BioLegend). […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Mus musculus
Diseases Basal Ganglia Diseases, Wounds and Injuries