Cyan adp flow cytometry analyzer

Part of isolated brain ECs were blocked with anti-Fc CD16/32 (1:100, BD Biosciences) before immunostaining with fluorescently labelled antibody cocktail specific for leukocyte/hematopoietic lineage marker CD45 (PE-conjugated; 1:100; eBiosciences) and the endothelial surface marker CD31 (APC-conjugated, 1:100, eBiosciences) or isotype control antibodies (APC and PE-conjugated, 1:100, eBiosciences). Cells were then analyzed by flow cytometry on the Beckman Coulter Cyan ADP Flow cytometry analyzer and Summit 4.3 software. Cell debris and dead cells were excluded from the analysis based on scatter signals.

A total of 30,000 events per sample were acquired in a CyAn ADP flow cytometry analyzer (Beckman Coulter, Inc., Brea, CA, USA). Data acquisition and analyses were performed using the Summit software 4.3.01 (Beckman Coulter, Inc., Brea, CA, USA). Distinct gate strategies, as previously described by Vitelli-Avelar et al. [16 (link)], were applied for data analysis using the FlowJo software (version 9.4.1, TreeStar Inc. Ashland, OR, USA).