PMID- 9683633
OWN - NLM
STAT- MEDLINE
DCOM- 19981009
LR  - 20220408
IS  - 0021-9533 (Print)
IS  - 0021-9533 (Linking)
VI  - 111 ( Pt 16)
DP  - 1998 Aug
TI  - Stimulated release of fluorescently labeled IgE fragments that efficiently 
      accumulate in secretory granules after endocytosis in RBL-2H3 mast cells.
PG  - 2385-96
AB  - Sensitization of RBL-2H3 mast cells with monomeric fluorescein-5-isothiocyanate 
      (FITC)-labeled immunoglobulin E (IgE) results in slow but highly efficient 
      accumulation of labeled IgE fragments in a pool of acidic peripheral vesicles 
      that are visible by fluorescence microscopy after raising endosomal pH with 
      ammonium chloride. Stimulation of cells containing these FITC-IgE fragments by 
      aggregation of high affinity receptors for IgE (FcepsilonRI) or by Ca2+ ionophore 
      and phorbol 12-myristate 13-acetate results in release of FITC fluorescence from 
      the cells, which can be monitored continuously with a spectrofluorometer. The 
      fluorescence release process corresponds to cellular degranulation: it is 
      prevented under conditions that prevent stimulated beta-hexosaminidase release, 
      and these two processes exhibit the same antigen dose-dependence and kinetics. 
      Pulse-chase labeling reveals that aggregation of FITC-IgE bound to FcepsilonRI at 
      the cell surface causes internalization and delivery to the regulated secretory 
      vesicles with a high efficiency similar to monomeric IgE-FcepsilonRI, but more 
      rapidly. Binding of Cy3-modified IgE to FcepsilonRI results in labeling of the 
      same secretory vesicles as in FITC-IgE-sensitized cells, and these Cy3-labeled 
      vesicles can be observed by fluorescence microscopy without neutralization of 
      intracellular compartments. Simultaneous three-photon microscopy of serotonin 
      fluorescence and two-photon microscopy of Cy3 fluorescence reveals that these 
      Cy3-labeled vesicles coincide with serotonin-labeled secretory granules. After 
      stimulation of the cells via aggregation of IgE-FcepsilonRI or addition of Ca2+ 
      ionophore and phorbol 12-myristate 13-acetate, depletion of the Cy3 label from 
      the intracellular vesicles is observed with confocal microscopy. These results 
      provide strong evidence for the lysosomal nature of secretory granules in these 
      cells. In addition, they provide the basis for a direct, real-time method for 
      monitoring single cell degranulation.
FAU - Xu, K
AU  - Xu K
AD  - Department of Chemistry, Cornell University, Ithaca, NY, USA.
FAU - Williams, R M
AU  - Williams RM
FAU - Holowka, D
AU  - Holowka D
FAU - Baird, B
AU  - Baird B
LA  - eng
GR  - AI18306/AI/NIAID NIH HHS/United States
GR  - GM08267/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - England
TA  - J Cell Sci
JT  - Journal of cell science
JID - 0052457
RN  - 0 (Carbocyanines)
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Immunoglobulin Fragments)
RN  - 0 (Ionophores)
RN  - 0 (Receptors, IgE)
RN  - 0 (cyanine dye 3)
RN  - 333DO1RDJY (Serotonin)
RN  - 37341-29-0 (Immunoglobulin E)
RN  - I223NX31W9 (Fluorescein-5-isothiocyanate)
RN  - NI40JAQ945 (Tetradecanoylphorbol Acetate)
SB  - IM
MH  - Animals
MH  - Carbocyanines
MH  - Cell Degranulation/drug effects
MH  - Cytoplasmic Granules/immunology/physiology
MH  - Endocytosis
MH  - Exocytosis
MH  - Fluorescein-5-isothiocyanate
MH  - Fluorescent Dyes
MH  - Immunoglobulin E/*physiology
MH  - Immunoglobulin Fragments/*physiology
MH  - Ionophores/pharmacology
MH  - Mast Cells/drug effects/*immunology/*physiology
MH  - Microscopy, Confocal
MH  - Microscopy, Fluorescence
MH  - Rats
MH  - Receptors, IgE/metabolism
MH  - Serotonin/metabolism
MH  - Tetradecanoylphorbol Acetate/pharmacology
EDAT- 1998/07/31 00:00
MHDA- 1998/07/31 00:01
CRDT- 1998/07/31 00:00
PHST- 1998/07/31 00:00 [pubmed]
PHST- 1998/07/31 00:01 [medline]
PHST- 1998/07/31 00:00 [entrez]
AID - 10.1242/jcs.111.16.2385 [doi]
PST - ppublish
SO  - J Cell Sci. 1998 Aug;111 ( Pt 16):2385-96. doi: 10.1242/jcs.111.16.2385.