Dears, I perform B cell Elispot on mouse splenocytes to measure the number of antigen-specific plasmablasts 7-8 days after immunization with a protein antigen. I need your help to understand why my Elispot wells are so dirty (see the attached figure) after development. I use a common protcol that here I summarize:
1- coating ON 4° with the antigen or control (non-related antigen, I use BSA)
2- Blocking, 3h RT with PBS+10%FCS
3- I plate splenocyets at 800 000 cells/well and perform serial dilutions
4- incubation 3h 37°
4- wash plate 3 times with PBS
5- wash plate 3 times with PBS+Tween 0.05%
6- secondary antibody and wash
7- HRP and wash
8- AEC Kit and development.
As you can observe in the picture, at the highest dilution of cells (800 000 cells/well) I have very dirty wells that do not allow me to count the spots. This dirty decrease with the number of cells and is not observed when I perform the same protocol to detect Bone Marrow Plasmacells and Memory B cell Elsipot. The dirty is observed also in BSA coated wells.
I think it's something related to the mouse spleen that I'm not able to wash away. Does anyone has some suggestion about why it happen or a possible solution (different washing buffers)?
Thank you very much,