Characterization of viable autofluorescent macrophages among cultured peripheral blood mononuclear cells

Background: Inflammatory macrophages that demonstrate intense autofluoresce nce have been isolated directly from alveolar and peritoneal tissues, but t heir generation in vitro remains vague. We use flow cytometry to identify a population of autofluorescent macrophages as they arise among nonadherent populations of cultured blood mononuclear cells. Methods: Cells R ere obtained from donated blood buffy coats and placed in culture for ii days. Unstained populations from the cells remaining in susp ension were sampled daily using flow cytometry. During the first 5 culture days, a distinct population of autofluorescent cells arose and comprised an avenge of less than or equal to 14% of the total cell population. This pop ulation declined to less than 6% by culture day 8. Results: The cells were identified as viable macrophages expressing CD68, l ysozyme, and HLA-DR. Quantitative reverse transcription-polymerase chain re action (RT-PCR) demonstrated a unique cytokine profile with IL-1 alpha expr ession levels 138-fold higher than those measured in uncultured monocytes. No significant elevation in the levels of other cytokines was identified. U pon replating, the sorted populations became readherent, were able to inges t plastic beads, and remained viable for G or more additional weeks in cult ure without evidence of proliferation or multinucleation. Conclusion: Viable autofluorescent macrophage populations arising among cul tured peripheral blood may be easily identified and isolated for further st udy using flow cytometry. Cytometry 44:38-44, 2001. Published 2001 Wiley-Li ss, Inc.(dagger).