Characterization of viable autofluorescent macrophages among cultured peripheral blood mononuclear cells.
BACKGROUND
Inflammatory macrophages that demonstrate intense autofluorescence have been isolated directly from alveolar and peritoneal tissues, but their 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 were obtained from donated blood buffy coats and placed in culture for 14 days. Unstained populations from the cells remaining in suspension were sampled daily using flow cytometry. During the first 5 culture days, a distinct population of autofluorescent cells arose and comprised an average of < or =14% of the total cell population. This population declined to less than 6% by culture day 8.


RESULTS
The cells were identified as viable macrophages expressing CD68, lysozyme, and HLA-DR. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) demonstrated a unique cytokine profile with IL-1 alpha expression levels 138-fold higher than those measured in uncultured monocytes. No significant elevation in the levels of other cytokines was identified. Upon replating, the sorted populations became readherent, were able to ingest plastic beads, and remained viable for 6 or more additional weeks in culture without evidence of proliferation or multinucleation.


CONCLUSIONS
Viable autofluorescent macrophage populations arising among cultured peripheral blood may be easily identified and isolated for further study using flow cytometry. Cytometry 44:38-44, 2001. Published 2001 Wiley-Liss, Inc.
Njoroge
J M
JM
Laboratory of Chemical Biology, NIDDK, National Institutes of Health, Bethesda, Maryland, 20892, USA.
Mitchell
L B
LB
Centola
M
M
Kastner
D
D
Raffeld
M
M
Miller
J L
JL
D002454:Cell Differentiation; D002470:Cell Survival; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology; D008264:Macrophages / Q000166:cytology* / Q000276:immunology
D005456:Fluorescent Dyes; D016650:Fluorescein-5-isothiocyanate