After 8 days of culture (T8), adherent fibrocytes were lifted by incubation in ice-cold 0.05% EDTA in phosphate-buffered saline (PBS), and cell viability was determined by the trypan blue exclusion test.
Characterization and identification of fibrocytes was performed at T0 and T8 by FACS (Beckman Coulter Company) using anti-CD45 (anti-CD45-krome orange, Beckman Coulter Company), anti-COL I (anti-COL I-FITC, Milli-Mark, Millipore), anti-CXCR4 (anti-CXCR4-PE, Beckman Coulter Company), anti-CD14, anti-CD86, and anti-HLA-DRII monoclonal antibodies (anti-CD14-alexa Fluor 750, anti-CD86-PC7, and anti-HLA-DRII-PC5.5, Beckman Coulter Company) [30 (link)].
Relevant isotype controls for each monoclonal antibody were used in the initial setup and frequently between tests.
Characterization and identification of fibrocytes was performed at T0 and T8 by FACS (Beckman Coulter Company) using anti-CD45 (anti-CD45-krome orange, Beckman Coulter Company), anti-COL I (anti-COL I-FITC, Milli-Mark, Millipore), anti-CXCR4 (anti-CXCR4-PE, Beckman Coulter Company), anti-CD14, anti-CD86, and anti-HLA-DRII monoclonal antibodies (anti-CD14-alexa Fluor 750, anti-CD86-PC7, and anti-HLA-DRII-PC5.5, Beckman Coulter Company) [30 (link)].
Relevant isotype controls for each monoclonal antibody were used in the initial setup and frequently between tests.
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