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EXPERIMENTAL EXAMPLE 1-1: Inhibition of Degranulation of Mast Cells - ¦Â-Hexosaminidase Assay
The rat basophilic leukemia cell line RBL-2H3 was seeded at a density of 2x105 cells/well in MEM supplemented with 15% FBS and 100 units/ml penicillin and streptomycin and incubated for 24 hours at 37ºC in a 5% CO2 atmosphere.
After removal of the medium, the cells were incubated for 4 hours in a fresh MEM medium containing 25 ng/ml anti-DNP IgE.  Then, the cells were washed with PIPES buffer (25mM PIPES, 119 mM NaCl, 5 mM KCl, 1 mM CaCl2, 0.4 mM MgCl2, 40 mM NaOH, 5.6 mM glucose, 0.1% BSA) before pre-incubation for 10 min.  Thereafter, the cells were treated for 20 min with 25, 50 and 100 ¦Ìg/ml the samples of Example 1 and then for 20 min with 50 ng/ml DNP-BSA.  The reaction was terminated at 4ºC and the supernatant was plated in an amount of 25 ¦ÌL/well into 96-well plates and reacted with 25 ¦ÌL of  1 mM P-nitrophenyl-acetyl-¦Â-D-glucosaminide at 37ºC for 1 hour in each well. The reaction was terminated with 0.1 M citrate buffer before absorbance at 405 nm was read in an ELISA reader.

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