Responses during cell preparation for functional analyses in mouse bone marrow-derived cultured mast cells.
Murine bone marrow-derived cultured mast cells (BMMCs) are most widely used in in vitro experiments for evaluation of mast cell functions. The present study has shown that cell preparation procedure, i.e., cell collection by centrifugation and the subsequent adjustment and culture of cell density at the desired concentrations, transiently induced gene expression of plasminogen activator inhibitor-1 (PAI-1) and the AP-1 components (c-fos, c-jun, and junB). The level of PAI-1 gene transcript was closely related to the cell density and the gene expression was enhanced by pretreatment with okadaic acid, an inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A). The cell preparation procedure also caused dephosphorylation of MAP kinases, i.e., ERK, p38, and JNK, resulting from PP1/PP2A activation. In view of the cell responses to the cell preparation procedure itself, care is needed in the interpretation of in vitro data using BMMCs.
Laboratory of Molecular Biology, Azabu University School of Veterinary Medicine, Sagamihara, Japan.
Masaru
M
Murakami
Teruo
T
Ikeda
Yoshii
Y
Nishino
Masayuki
M
Funaba
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000201:enzymology / Q000276:immunology / Q000502:physiology; D002452:Cell Count; D018929:Cell Culture Techniques; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D008407:Mast Cells / Q000166:cytology / Q000201:enzymology / Q000276:immunology / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D020928:Mitogen-Activated Protein Kinases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000235:genetics; D054648:Protein Phosphatase 2; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018808:Transcription Factor AP-1 / Q000096:biosynthesis* / Q000235:genetics
D004791:Enzyme Inhibitors; D017395:Plasminogen Activator Inhibitor 1; D018808:Transcription Factor AP-1; D019319:Okadaic Acid; D020928:Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; C117094:Ppp2r3a protein, mouse; D054648:Protein Phosphatase 2