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Guest Michelle

technical support B-cell IgM

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Guest Michelle

 We are trying to set up a B-cell Elispot assay for IgM. but  are facing some  new challenge, deciding which spots are legit and which are false, setting our protocol boundaries spots size wise.

We were hoping someone could help with that decision.

For us it is unclear what size spots you can expect for IgM. But also the differences in spot size in unstimulated and stimulated wells is notable.

Does one of you have a answer?

Thanks!

 

IgM Elispot.pptx

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Dear Michelle, 

Welcome to our forum!

 

When it comes to IgM I really believe that all spots you are seeing in unstimulated controls are "real" spots. They are not artefacts. However, there is a clear stimulation increase and we want to set our counting paramters in a way so that this increase in the number of bigger spots is accurtaly captured. 

For me this is about making a research decision. Pick a count setting and then stick to it throughout your study, given that all donors and plates have been handeled in the same way! Evalute 3-5 donors before making a decision on which setting is good. If plates have not been developed equal times with substrate having the same count-setting could be impossible. 

Excluding "real" spots like this is in my mind totally fine, although I prefer If the "thresholding" is done in a consisten manner on all plates due to the study having been performed in the same way over and over again (substrate development time being crucial). The important part is that you should be able to send your plates to a collaborator in United States with the same type of reader. He should then be able to use the same count settings and extract the same spot counts from your plates within +-3%. 

I have taken your PPT and shown with arrow which kind of spots I would want to count in your unstimulated controls. I have done this pretty quickly so dont take it litteraly. But you can see that I would want you to use a count setting threshold which focuses on counting the bigger spots. Exclude the small unspecific background. In your example you will get a very nice stimulation index. Does all your IgM ELISpot plates look this good? Great job.  

 

IgM feedback.pptx

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