PMID- 10404150
OWN - NLM
STAT- MEDLINE
DCOM- 19990831
LR  - 20191103
IS  - 0196-4763 (Print)
IS  - 0196-4763 (Linking)
VI  - 36
IP  - 4
DP  - 1999 Aug 1
TI  - Quantitative measurement of mast cell degranulation using a novel flow cytometric 
      annexin-V binding assay.
PG  - 340-8
AB  - BACKGROUND: Mast cells are primary mediators of allergic inflammation. 
      Antigen-mediated crosslinking of their cell surface immunoglobulin E (IgE) 
      receptors results in degranulation and the release of proinflammatory mediators 
      including histamine, tumor necrosis factor-alpha, and leukotrienes. METHODS: Mast 
      cells were stimulated to degranulate by using either IgE crosslinking or 
      ionophore treatment. Exogenously added annexin-V was used to stain exocytosing 
      granules, and the extent of binding was measured flow cytometrically. Release of 
      the enzyme beta-hexosaminidase was used for population-based measurements of 
      degranulation. Two known inhibitors of degranulation, the phosphatidylinositol 3 
      kinase inhibitor wortmannin and overexpression of a mutant rab3d protein, were 
      used as controls to validate the annexin-V binding assay. RESULTS: Annexin-V 
      specifically bound to mast cell granules exposed after stimulation in proportion 
      to the extent of degranulation. Annexin-V binding was calcium dependent and was 
      blocked by phosphatidylserine containing liposomes, consistent with specific 
      binding to this membrane lipid. Visualization of annexin-V staining showed 
      granular cell surface patches that colocalized with the exocytic granule marker 
      VAMP-green fluorescent protein (GFP). Wortmannin inhibited both annexin-V binding 
      and beta-hexosaminidase release in RBL-2H3 cells, as did the expression of a 
      dominant negative rab3d mutant protein. CONCLUSIONS: The annexin-V binding assay 
      represents a powerful new flow cytometric method to monitor mast cell 
      degranulation for functional analysis.
CI  - Copyright 1999 Wiley-Liss, Inc.
FAU - Demo, S D
AU  - Demo SD
AD  - Rigel Inc., South San Francisco, California, USA.
FAU - Masuda, E
AU  - Masuda E
FAU - Rossi, A B
AU  - Rossi AB
FAU - Throndset, B T
AU  - Throndset BT
FAU - Gerard, A L
AU  - Gerard AL
FAU - Chan, E H
AU  - Chan EH
FAU - Armstrong, R J
AU  - Armstrong RJ
FAU - Fox, B P
AU  - Fox BP
FAU - Lorens, J B
AU  - Lorens JB
FAU - Payan, D G
AU  - Payan DG
FAU - Scheller, R H
AU  - Scheller RH
FAU - Fisher, J M
AU  - Fisher JM
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Cytometry
JT  - Cytometry
JID - 8102328
RN  - 0 (Androstadienes)
RN  - 0 (Annexin A5)
RN  - 0 (Liposomes)
RN  - 0 (Luminescent Proteins)
RN  - 0 (Phosphatidylserines)
RN  - 147336-22-9 (Green Fluorescent Proteins)
RN  - EC 3.2.1.52 (beta-N-Acetylhexosaminidases)
RN  - EC 3.6.1.- (GTP-Binding Proteins)
RN  - EC 3.6.5.2 (rab3 GTP-Binding Proteins)
RN  - SY7Q814VUP (Calcium)
RN  - XVA4O219QW (Wortmannin)
SB  - IM
MH  - Androstadienes/pharmacology
MH  - Animals
MH  - *Annexin A5/metabolism
MH  - Calcium/pharmacology
MH  - *Cell Degranulation/drug effects
MH  - Cytoplasmic Granules/metabolism
MH  - Flow Cytometry/*methods
MH  - GTP-Binding Proteins/genetics/metabolism
MH  - Green Fluorescent Proteins
MH  - Liposomes/pharmacology
MH  - Luminescent Proteins
MH  - Mast Cells/*physiology
MH  - Mice
MH  - Microscopy, Fluorescence
MH  - Phosphatidylserines/pharmacology
MH  - Protein Binding/drug effects
MH  - Wortmannin
MH  - beta-N-Acetylhexosaminidases/analysis
MH  - rab3 GTP-Binding Proteins
EDAT- 1999/07/15 00:00
MHDA- 1999/07/15 00:01
CRDT- 1999/07/15 00:00
PHST- 1999/07/15 00:00 [pubmed]
PHST- 1999/07/15 00:01 [medline]
PHST- 1999/07/15 00:00 [entrez]
AID - 10.1002/(SICI)1097-0320(19990801)36:4<340::AID-CYTO9>3.0.CO;2-C [pii]
AID - 10.1002/(sici)1097-0320(19990801)36:4<340::aid-cyto9>3.0.co;2-c [doi]
PST - ppublish
SO  - Cytometry. 1999 Aug 1;36(4):340-8. doi: 
      10.1002/(sici)1097-0320(19990801)36:4<340::aid-cyto9>3.0.co;2-c.