We are conducting an active measurement experiment of Beta-hexosaminidase (B-hexo) through RBL-2H3 cells.
The courses are as follows:
1. Seed RBL-2H3 cells to 96 well plates at 500 ng/ml with a final concentration of anti-DNP-IgE of 8 x 10^4/well and incubate for a day.
2. Remove the top liquid and wash it twice with DPBS.
3. 2 hr pre incubation with Siraganian buffer
4. Remove the supermatamt and culture the DNP-BSA dissolved in the Siraganian buffer for 30 minutes.
(Final concentration: 150ng/ml)
5. Transfer 60ul of the supernatant to a new 96 well plate and react 3 hr at 37°C with a 10mM
P-NAG (in 0.1M Citrate buffer) of 40ul.
6. Stop the reaction with 0.05M sodium carbonate 100ul and measure absorbance at 405nm.
However, no B-hexo was created in positive control.
What's wrong with my process?