Rabbit IFN-r ELISPOT-Abnormal dark background

[Guest Sherry]

Hi MabTech, I ran a rabbit IFN-r ELISPOT kit with rabbit PBMCs. My positive controls are row G and H, negative controls are row A, B, E, and F, and my experiments are row C, D. Most of the wells work pretty well, while some wells have abnormal super dark background, specifically C2, C3, D1, D2, D3, E2. Because of this high background, the ELISPOT reader couldn't count the spots in these wells. May I know your advice how to improve? Thanks!

[Christian@mabtech.com]

Dear Sherry,

Darkened membranes can have many, many causes but looking at D1 and B2 I seem to see "a drying out" membrane. During the entire assay it is important to keep the membrane hydrated with PBS as much as possible. You obviously have to empty the wells at some points but liquid should quickly be added, otherwise you run the risk of drying out the membrane. The air will eventually begin to dry out the PVDF and the beginning stages of this often looks "patchy" very similar what is observed in well D1 and B2. 

The early stages of drying changes the PVDF membrane causing following detections steps to react differently in such wells. 

Drying can happen at:

coating steps

Cell seeding steps

Following removal of cells and all detection steps. 

 

[Guest Sherry]

Thank you so much for the reply!