Image analysis of mast cell degranulation in a concentration gradient of stimuli formed in the channel between a glass plate and a silicon substrate.
Measurement of released granule components, popularly used to quantify mast cell exocytosis, does not deliver real-time information about degranulation at the single-cell level nor the ratio of responding/non-responding cells. Rather it provides, only end-point, bulk-population data. Here we studied degranulation of rat peritoneal mast cells dispersed in a narrow horizontal channel between a silicon substrate and a glass plate. Upon exposure to a concentration gradient of a soluble stimulus, degranulation started from those cells facing towards the highest concentration of stimulus. We captured images of exocytosing cells without the need for phase-contrast or differential interference-contrast microscopy. This was achieved using the reflection caused by the silicon substrate. The time-lapse images of cells in the channel were segmented into multiple concentration belts to identify the proportion of degranulated cells in each belt region. Maximum ratios of degranulated cells in the belt regions determined by time-course curve fitting calculations were then plotted against the distance from the stimulus injection site, resulting in a sigmoidal response curve. This method provides a powerful means for real-time analysis of concentration- and stimulus-dependent degranulation of mast cells and allows comparison of cell responses under different conditions. To show its effectiveness, we evaluated the effect of a protein kinase C (PKC) inhibitor, Gö6976, on degranulation induced by various stimuli. In contrast to stimulation with concanavalin A+lysophosphatidylserine (lysoPS) or nerve growth factor+lysoPS (completely inhibited by Gö6976 over the whole range of stimulus concentrations used) or compound 48/80 and mastoparan (no inhibition by Gö6976), stimulation with ionomycin, a known Ca(2+) ionophore, showed a concentration-dependent inhibition by Gö6976, with a major inhibition at low stimulus concentrations and a diminished one at higher ionomycin concentrations. The results indicate that ionomycin-induced degranulation is mainly induced via a PKC-independent signal cascade at high stimulus concentrations, whereas below a certain concentration, degranulation is completely dependent on PKC.
Nitta
Nao
N
Central Laboratoty, ECI Inc., Meguro-ku, Tokyo, Japan.
Aoki
Yoshiko
Y
Isogawa
Yasushi
Y
Tsuchiya
Tomoko
T
Kanegasaki
Shiro
S
D000818:Animals; D015550:Cell Degranulation; D005898:Glass; D007091:Image Processing, Computer-Assisted / Q000379:methods; D015759:Ionomycin / Q000494:pharmacology; D008407:Mast Cells / Q000276:immunology* / Q000648:ultrastructure*; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D012825:Silicon
D015759:Ionomycin; D011493:Protein Kinase C; D012825:Silicon