Is it possible to use the MultiScreen® Vacuum Manifold after plates have dried? Many of my wells have a darker background/membrane staining following a day of drying (see attachment). The vacuum manifold wasn't used initially. Would using it to re-wash the plates help, or could this harm the assay since the membranes are fairly dry since yesterday?
Hi,
I would advise against using a vaccum washer after development and drying is done. I don't think you will get the result you desire.
Darkening of membranes like this are often caused by massive number of spots in the affected wells or from a general membrane background due to poor cell viability (alot of apoptotic cells).
In the case that there are many many spots in the wells extra washing will not help. Titrating down cell numbers is a better approach.
If the darkening is to a general background there are few things to consider:
- Low cell viability will have many apopotic cells and debris in it. These will stick to the membrane during the assay and will make the detection antibodies get trapped. Most of this debri end up in the outer periphery of the well so you get the charaterstic "ring formation". Best fix for this is ofcouse improve the viability of the analyzed cells. You can also try more rigourous wash protocols before addition of the first detection mab. First 5x washes with PBS, then incubating the plates with 100ul/well of 2mM EDTA in PBS for 10minutes in 37C, and then an additional 5x washes with PBS. This can reduce the amount of debri stuck on the membrane but it is no magical bullet. It can reduce the background, not remove it.
- Some peptides and antigens are known to generate much darker ELISpot membranes. There are speculations that antigens can make the cells go abit crazy spewing out something sticky, but it could also that some peptides have a polarity that really sticks to the PVDF membrane. In both cases there is little to do than adjust development time with substrate so you still see the spots despite darkening membranes.
- Dark membranes can be caused by human AB serum that contains heterophilic antibodies. They can crossbind the coating and detection mabs in the assay. We therefore never recommend using human AB serum in ELISpot.