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Evidence for uptake of vital dye by activated rat peritoneal mast cells: an in vitro imaging study
Abstract:
Uptake of material from surrounding medium by activated rat peritoneal mast cells (PMCs) was studied using in vitro peritoneal eluate cells, the vital fluorescent dye sulforhodamine B (SFRM-B), secretagogue compound 48/80, and an imaging technique. PMCs, which undergo different states of degranulation, are shown to possess the ability to take up (by endocytosis) SFRM-B in an activity-dependent manner. The endocytosed dye is incorporated in the granules and can be discharged into the medium when the cells are reactivated. Both the uptake and the discharge processes are calcium-dependent. The reactivity of mast cells to secretagogue is not altered by the application of the dye. SFRM-B, a negatively charged, nonspecific protein stain, displays greater photostability and less leakage than the positively charged acridine orange, and its fluorescence persists for hours, whereas acridine orange fluorescence fades within 1 min when exposed to ultraviolet illumination. The fluorescent image of the dye-loaded mast cells can be preserved overnight in a container at room temperature. SFRM-B elicits no detectable damaging influence on the activated afferent discharge of splanchnic afferent nerve fibers with mesenteric terminals. This enables the use of SFRM-B for studying the interactions between mesenteric afferent terminals and their surrounding mast cells.
Insights
Activated rat mast cells can internalize and release the dye sulforhodamine B (SFRM-B) through an endocytosis process. This calcium-dependent mechanism is vital for studying mast cell interactions with nerve fibers.
Area of Science:
- Cell Biology
- Neuroscience
- Immunology
Background:
- Rat peritoneal mast cells (PMCs) play a role in cellular signaling and immune responses.
- Understanding mast cell degranulation and material uptake is crucial for studying cellular interactions.
- Previous methods for tracking mast cell activity had limitations, such as dye photobleaching and leakage.
Purpose of the Study:
- To investigate the uptake and release mechanisms of the fluorescent dye sulforhodamine B (SFRM-B) by activated rat mast cells.
- To evaluate SFRM-B as a tracking tool for mast cell activity and its interactions with nerve fibers.
- To compare SFRM-B with other fluorescent dyes like acridine orange for mast cell imaging.
Main Methods:
- Utilized in vitro rat peritoneal eluate cells and the vital fluorescent dye SFRM-B.
- Stimulated mast cell degranulation using secretagogue compound 48/80.
- Employed an imaging technique to observe dye uptake and release, alongside calcium dependency studies.
- Assessed the dye's photostability, leakage, and effects on splanchnic afferent nerve fibers.
Main Results:
- Activated PMCs demonstrated an activity-dependent uptake of SFRM-B via endocytosis.
- Endocytosed SFRM-B was incorporated into granules and could be discharged upon cell reactivation.
- Both uptake and discharge processes were calcium-dependent.
- SFRM-B exhibited superior photostability and reduced leakage compared to acridine orange.
- SFRM-B did not adversely affect the activity of splanchnic afferent nerve fibers.
Conclusions:
- SFRM-B is effectively taken up and released by activated mast cells through an endocytosis mechanism.
- The dye's properties make it a suitable and stable fluorescent marker for studying mast cell behavior.
- SFRM-B facilitates research into the interactions between mast cells and mesenteric afferent nerve terminals without causing damage.