Protocol for Beta-Hexosaminidase RBL-2H3 Secretion Assay
1) Plate cells such that they are close to confluent when the assay is to be carried out
(in a 48-well plate, 2.5 x 105 cells/well; Fisher catalog number 07-200-86).
2) Wait at least 3 hours after plating before carrying out experiment (up to
overnight).
3) Sensitize the cells 3 hours to overnight with 1.0ug/ml IgE.
4) To remove excess IgE before stimulation, wash cells 4 times with extracellular
buffer with hepes.
5) To stimulate, add 800ng/ml DNP-BSA suspended in extracellular buffer with
0.1% BSA. The total volume of the supernatant should be 500ul. Incubate at
37C for 1hr.
6) In a control well, add 200nM ionomycin and 50nM PMA for 1hr at 37C.
7) Following incubation, take 50ul of the supernatant and incubate it with 200ul of
1mM p-nitrophenyl N-acetyl-beta-D-glucosamine in 0.05M citrate buffer (pH 4.5)
for 1hr at 37C.
8) As a control for total beta-hexosaminidase concentration, lyse cells with 1%
Triton X-100 and remove 50ul of the supernatant; incubate as in step 6.
9) Quench both reactions following incubation by adding 500ul of 0.05M sodium
carbonate buffer (pH 10.0).
10) Read OD of each reaction at 405nm.
Making Citrate Buffer:
1) Add 49.5mL of 0.05M citric acid to 50.5 of 0.05M trisodium citrate.
2) Check to see that pH is at 4.5.
Making Sodium Carbonate Buffer:
1) Add 60mL Na2CO3 to 40mL NaHCO3.
2) Check to see that pH is at 10.0.