Dear Pavel, welcome to the forum!
General background staining of the membrane typically can be traced back to these problems:
- The use of Human AB serum in the cell culture medium. Human serum can in some donors contain heterophilic antibodies which cross-links the capture and detection antibody.
- The use of Tween in the PBS when washing the plates. Tween is never recommended if you use Mabtech pre-coated plates or coat yourself but use EtOH preactivation.
- If you activate your cells prior to adding them into the elispot wells, the cell supernatant can contain large quantities of the cytokine analyzed, resulting in a general darkening of the membrane. I know from experience with human cells that IFNg secretion begins just around 30min after the cells have been mixed with peptides. As a result, if you mix cells and stimuli, then prepare your elispot plate and finally add cells+stimuli 40 min later, the end result could high backgrounds.
- Sometimes cell can be pre-activated in vivo. As a result, the splenocytes could have been actively producing IFNg at the time the cells were frozen. This continues even after thawing of the cells. During the 1h post thawing, IFNg will be released into the cell tubes and you can end up with rather large amounts of free IFNg in your cell solution. This obviously then darkens the membrane. One way to get rid of this problem is to wash the cells right before adding them into the ELISpot plates.
- Some peptides are known to cause dark membrane staining. So do you see this darkening only for some peptides? Or do you see it in all wells of the plate? We believe this could be related to high concentration of DMSO, but other factors could be in play as well. High DMSO can cause the membrane to leak and detection antibodies go deeper into the PVDF membrane. The result is strong membrane darkening. One should not use higher than 0.4% DMSO. In case your peptide needs high DMSO to be soluble, just dissolve the peptide in a small amount high concentration DMSO. Then dilute it down in PBS. The peptide will remain soluble even below 0.4%. What you also can do is lower concentration of the peptide down to 1ug/ml. That most often works just as well as 5ug/ml.
So, questions: Are you using Tween? Possible you have invivo activated splenocytes? Is the darkening of the membrane happening all wells, or just in some peptides activated wells?
best,
Christian
