Simple screening method for inhibitory effects of food ingredients on degranulation in RBL-2H3 cells

RBL-2H3 rat mast cells were stimulated to secrete intracellular granules through multivalent binding of antigen to receptor-bound IgE and preferentially used for anti-allergic evaluation. beta-Hexosaminidase stored in the granules was used as a marker of mast cell degranulation. We developed a simpl...

Full description

Saved in:
Bibliographic Details
Published inNihon Shokuhin Kagaku Kōgaku kaishi Vol. 55; no. 11
Main Authors Horigome, S.(Japan Food Research Labs., Ibaraki, Osaka), Yoshida, I, Tamaki, C, Yamaguchi, A, Kibune, N, Kamibe, T, Watai, M
Format Journal Article
LanguageJapanese
Published 01.01.2008
Subjects
ALIMENTOS
ENZIMAS
ENZYME
ENZYMES
FONCTION PHYSIOLOGIQUE
FOODS
FUNCION FISIOLOGICA
HYPERSENSITIVITY
PHYSIOLOGICAL FUNCTIONS
PRODUIT ALIMENTAIRE
REACCIONES ALERGICAS
REACTION ALLERGIQUE
Online AccessGet more information

Cover

More Information
Summary:RBL-2H3 rat mast cells were stimulated to secrete intracellular granules through multivalent binding of antigen to receptor-bound IgE and preferentially used for anti-allergic evaluation. beta-Hexosaminidase stored in the granules was used as a marker of mast cell degranulation. We developed a simple screening method to evaluate inhibitory effects of mast cell degranulation by efficient measurements of the enzyme activity in a continuous 96-well format. The cells were inoculated into a 96-well plate (2.5 x 10E5/well) and cultured overnight. Anti-dinitrophenyl-IgE, food extracts and dinitrophenyl-human serum albumin were successively added and incubated for each optimized interval of time. The supernatant was transferred to another well, and the cells were lysed with a passive lysis buffer instead of conventional sonication. beta-Hexosaminidase activity in the supernatant and the cells was measured using a color formation substrate with a microplate reader. The percentage of degranulation was calculated from the ratio of the enzyme activity (supernatant/cells). The proposed method can be easily completed by a multi-channel pipetting operation and adapted for high-throughput screening (21 samples/plate, n=2) as compared to the conventional 24-well based method (3 samples/plate, n=2). Reproducibility was also improved from RSD 12.3% to 5.4-9.0% (within-run, n=4). The results agreed well with those of the previous method.
Bibliography:Q04
2009000352
ISSN:1341-027X
DOI:10.3136/nskkk.55.535