Journal: PLoS ONE

Article Title: The Inhibition of Mast Cell Activation of Radix Paeoniae alba Extraction Identified by TCRP Based and Conventional Cell Function Assay Systems

doi: 10.1371/journal.pone.0155930

Figure Lengend Snippet: (A-B) IgE-mediated TCRP comparison with cell-electrode impedance dynamics induced by adenosine or PMA. RBL-2H3 cells were seeded on the E-plates overnight at a density of 20,000 cells/well, and were then treated with DNP-BSA (IgE-sensitized) and adenosine or PMA (no sensitization). (C) Representative distribution of CD63 stimulated by different antigens in RBL-2H3. RBL-2H3 cells with or without IgE sensitization were stimulated with DNP-BSA, adenosine, and PMA for 30 min. Bar, 5 μm. (D) Adenosine induced microtubule formation whereas PMA induced F-actin disassembly. Treated cells were fixed and processed for staining with phalloidin-rhodamine (red) and antibody to β-tubulin (green). Bar, 5 μm. (E) Adenosine together with PMA induced β-hexosaminidase release. The cells were seeded in 24-well plates overnight, pretreated with PMA or adenosine and subsequently activated with different concentrations of adenosine or PMA. *** P < 0.001 vs. with PMA or adenosine only.

Article Snippet: The cells were permeabilized in phosphate-buffered saline (PBS) containing 0.5% Triton X-100 for 15 min and blocked with PBS containing 1% BSA for 1 h. The following primary and secondary stainings were performed: CD63 antibody (Epitomics, Inc.; Burlingame, CA) at a dilution of 1:50, fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (MultiSciences Biotech Co.; Hangzhou, China) at 1:100, FITC-conjugated tubulin antibody (Cell Signaling Technology; Beverly, MA), phalloidin-rhodamine (Invitrogen Molecular Probes; Carlsbad, CA), and 4′6-diamidino-2-phenylindole (DAPI; Invitrogen).

Techniques: Staining