Hi Samira, yes there is colour in my cell lysates, which develops after the addition of glycine buffer.

It sounds like your cells lost response to stimuli. it could be happened to cells with high passage number or lacking of calcium or magnesium in your release buffer.

Hi Laurie, I've tried using a younger passage of cells and it's still the same.

I think the problem might be due to the lack of cells. I noticed that after the change of medium or sensitising the cells with IgE the following day (after seeding), a lot of my RBL cells have detached. 

This is my procedure:

Day 1 - Seed 4 x 104 cells/well with 5% EMEM (cells were previously cultured in 10% EMEM in T75 flask)

Day 2 - Cells are confluent. Change fresh medium (5% EMEm, for normal group) and sensitise cells with 1 microgram/ml of IgE diluted in 5% EMEM (for induced group) overnight.

Day 3 - Some cells have detached. :(

Urgently need help. Thanks.

The number of cells in does affecting the colour of the assay at the end, however it depends the percentage of release you normally get from the stimulus and how it compared to the cells detached.  I used a range of cell lysates to see if  the cells in the wells were optimized and used the cell in the top linear region of the curve and the spontaneously release were not too high as well. You can check the percentage of cell detached when compare lysate with and without IgE sensitization in the same experiment.

 Thank you for the inputs Laurie and Samira. Will definitely be trying out these suggestions. Will be optimising with reduced cell numbers next too. This problem did not happened to my seniors, totally baffled everyone.

Laurie, when you mentioned release buffer that is lacking Ca or Mg, do you mean Tyrode's buffer? My cells are induced with anti DNP-BSA prepared in Tyrode's buffer.

I do mean Tyrode's buffer.

Thanks Laurie. I tried to half the volume of Tyrode's buffer and there was colour development though faint. Do you suggest that I increase the concentration of anti DNP-BSA? Currently using 1 microgram/ml which most journals are using.

Dear Audrey, I will do a dose response of DNP-BSA from 0.1 microgram/ml to 10 microgram /ml, or see if you can increase the number of cell seed in the well. It also dependents what optical range your plate reader can handle . If the plate reader your using can handle O.D of 3 or above and the OD in your total lysate tube read just above O.D 1 you can try to used double amount of cell. Good luck.

Thanks Laurie, will definitely be doing that.