Optimized THP-1 differentiation is required for the detection of responses to weak stimuli.
OBJECTIVE
The differentiation of THP-1 monocytes into macrophages is mainly conducted at a phorbol 12-myristate 13-acetate (PMA) concentration of 10-400 ng/ml. However, this concentration might be high enough to upregulate the expressions of some genes in differentiated macrophages, which could overwhelm gene expression increases induced by other stimuli. The present study was performed to optimize the PMA concentration required to differentiate monocytes whilst minimizing gene upregulation.


METHODS
THP-1 cells were treated with 2.5-100 ng/ml PMA and analyzed for the extent of cell adherence, the surface marker of macrophages, and stable differentiation without undesirable gene upregulation. The stably differentiated THP-1 cells at the minimum PMA concentration were treated with 10 ng/ml LPS or 125 nM amyloid beta (Abeta(1-42)).


RESULTS
The treatment of THP-1 with 5 ng/ml PMA was found to be sufficient to induce stable differentiation without undesirable gene upregulation. These macrophages differentiated at 5 ng/ml responded well to secondary weak stimuli like 10 ng/ml LPS or 125 nM of amyloid beta (Abeta(1-42)).


CONCLUSIONS
This finding suggests that THP-1 cells are well differentiated by 5 ng/ml PMA, and that the resulting differentiated macrophages respond well to secondary weak stimuli without being overwhelmed by undesirable gene upregulation induced by PMA.
Park
E K
EK
East-West Bone and Joint Research Center, College of Medicine, Kyung Hee University, Seoul, South Korea.
Jung
H S
HS
Yang
H I
HI
Yoo
M C
MC
Kim
C
C
Kim
K S
KS
D016229:Amyloid beta-Peptides / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000502:physiology*; D009000:Monocytes / Q000187:drug effects*; D023282:Nuclease Protection Assays; D010446:Peptide Fragments / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects
D016229:Amyloid beta-Peptides; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D010446:Peptide Fragments; C075222:amyloid beta-protein (1-42); D013755:Tetradecanoylphorbol Acetate