I am happy to hear that your results improved by using our pre-coated plates! You are absolutely right, by having a kit that generates lower background, spot-definition is improved and the reader identifies spots easier. However, in many cases, it looks like there were more spots in the pre-coated plate compared to the manual plate. Could you send me a PowerPoint with images of pre-coated and manual coated wells taken from the original saved folder? I have given some instruction in the attached PPT. By doing it this way, I can look at the high definition version of the wells and can better give feedback.
1. Yes, a decrease in background will make it easier for the software to count the spots accurately. However, to me it seems like you actually have an overall more sensitive system using the pre-coated plate. Fundamentally more spots are detected using the pre-coated plate. However, I would have to see the high-resolution images to really say that.
2. You should get a feeling about changing the spot count settings on the AID reader. There is no right or wrong! The reader should be regarded as your unbiased partner in plate reading. If the reader is not counting 100% of the visual spots, only 70%, that is ok as long as you consistently use the same setting throughout the wells. You could for example exclude the weakest spots. In general, I think your setting looks good, although I would need to look at your high-resolution images to be absolutely sure.
3. Hard to say really. The flexible underdrain can lead to problems with leakage, which can dramatically affect the result of individual wells or section of the plate. However, it does not look like this is your issue as your suboptimal result for the manually coated plate seems consistent throughout all wells.
4. This is probably the main reason why you don’t see the same result in the manually coated plate. Mixing manufacturers is not recommended. Additionally, we make sure our streptavidin and substrate perform optimally for ELISpot.
The directly conjugated detection antibody 7-B6-ALP is a very reliable and good choice.
Save high resolution images.pptx