Guest Ellie Posted June 23, 2022 Report Share Posted June 23, 2022 Hi there! I have just developed an IFN-gamma ELIspot using the ELIspot Pro kit which has unfortunately resulted in dark membrane staining. I was just reading that this is sometimes caused by the addition of Tween 20 in the washing buffer. I did use Tween 20 in the wash buffer and was wondering if this darkness is a similar result to what you would expect when using this product? The wells pictured contained 200,000 cells with PHA - you can actually see spots when enough light is shone onto the plate. Thank you, Ellie Quote Link to comment Share on other sites More sharing options...
Christian@mabtech.com Posted June 23, 2022 Report Share Posted June 23, 2022 Hi, the darkness induced by tween does NOT look this. It is much more subtle effect on the membrane without any spots where you get a darkening compared to wells only washed with pure PBS. No, the issue you are having is most likely confluent spot formation. There are too many cells secreting IFNg so the whole membrane is blacked out. For PHA I recommend 50K cells/well for human IFNg. Are these precoated plates or have you coated yourself? Quote Link to comment Share on other sites More sharing options...
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