sebbeols Posted April 12, 2016 Report Share Posted April 12, 2016 Hey there Mabtech! I have a few questions about the B cell ELISpot in general and also some on how I should handle the analysis of my data. Plus a question on my sample material. General questions: According to U-Cytech's ELISpot protocol, they recommend that one washes the back of the membrane between the detection Ab step and the SA-enzyme step to reduce background staining. I have never tried this and find it a little odd, do you have any thoughts on this? With my B cell ELISpot I am studying the plasmablast response after vaccination in NHPs. I have found mixed opinions about the viability of plasmablasts after freeze/thawing, do you have any experience with this? Nakaya et al., Nat Immunol 2012, write that plasmablasts don't survive freeze/thawing but do not show any data to support this claim. I have yet to try running my B cell ELISpot for plasmablasts from frozen samples, but will do so soon. Do you recommend resting the cells o/n after freeze/thawing? Experiment specific: When I run my B cell ELISpot, I always make a 3-fold dilution down the plate (see attached image). To back-calculate the number of ASCs/million PBMCs, I have had a hard time finding guidance on which dilution well I should use. Is there a certain range of spots I should aim for? Should I be consistent with which column I choose to count from? Can I do an average from multiple dilution wells (i.e. columns)? Each sample is run in duplicates (2 rows). For my Ag-specific wells I coat them with our vaccinating antigen. I also include an irrelevant antigen (OVA) which I coat a well with. What should I do with the spots that get counted in the irrelevant antigen well? I use this negative control well to optimize the counting algorithm for the AID program, but I will never be able to eliminate all unspecific spots without also losing positive spots. Should I subtract the negative spots that are counted from the Ag-specific spots? Thanks in advance for your help and feedback! /Sebastian Link to comment Share on other sites More sharing options...
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