Rat basophilic leukemia mast cell line RBL-2H3 was obtained from the American Type Culture Collection (Manassas, VA, USA).
Rat RBL-2H3 mast cells were grown in MEM-[alpha] medium supplemented with 10% heat-inactivated FBS containing 1% antibiotics (ABS).
Here, vaccination with CL531 plus OVA significantly reduced mediator release from RBL-2H3
cells compared to PBS-vaccinated and OVAsensitized animals (PBS group).
We measured degranulation of RBL-2H3
mast cells by using [beta]-hexosaminidase assay.
Rat basophilic leukemia cells (RBL-2H3
) (ATCC, Rockville, MD, USA) were seeded on 6-well (1 x [10.sup.6] cells/well) plates and primed with anti-DNP IgE (200 ng/m1) overnight and then challenged with DNP-HSA (300 ng/m1) for 30 min for mast cell degranulation analysis.
In the study, we used two kinds of rat mast cells, rat basophilic leukemia (RBL-2H3
) cells and rat peritoneal mast cells (RPMCs).
They identified recombinant G ([beta]2r2) subunit markedly synergized PLD activation by compound 48/80 in permeabilized RBL-2H3
A colorimetric assay was performed to determine [beta]-hexosaminidase activity in RBL-2H3
cells using a [beta]-N-acetyl glucosaminidase (NAG) activity assay kit (Biovision, Zurich, Switzerland).
To determine the amount of [beta]-hexosaminidase activity released by BaaC or RBL-2H3
cells, 10 or 25[micro]l of supernatant and 90 or 50[micro]l of 1.2 ma [beta]-hexosaminidase substrate (4-nitrophenyl-N-acetyl-[beta]-D-glucosaminid; Sigma-Aldrich, Germany) in 0.1 a sodium acetate buffer (pH 4.5) were mixed in 96-well plates and incubated for 2 h at 37[degrees]C.
cells (American Type Culture Collection, Manassas, VA, USA) cultured on a 24-well plate were stimulated at 37[degrees]C under gentle rotation.
Since, [beta]-hexosaminidase is usually released along with histamine from mast cells or basophils, this enzyme is therefore used as the marker for mast cell degranulation in RBL-2H3
cell line (Cheong et al.
Previously, we reported that the structural requirements of the 3-phenylisocoumarins for inhibition of degranulation and/or release of TNF-[alpha] and IL-4 in rat basophilic leukemia (RBL-2H3
) cells (Wang et al., 2007).