Guest Rain Posted April 14 Report Share Posted April 14 Dear Mabtech support team I used mouse IFN-γ ELISpot kit, and I found that the spots are so blurring, like picture 1 and 2. Could you please tell me why could this happen? Thanks a lot. Looking forward to your suggestions! Thanks, Rain 1.bmp 2.bmp Quote Link to comment Share on other sites More sharing options...
Christian@mabtech.com Posted April 14 Report Share Posted April 14 The spots look rather large and blurry, yes. Is this a precoated kit you purchased from Mabtech or have you coated the plates yourself? What concentration of capture mab did you use? Are these mouse splenocytes incubated for a long time in the plate? Please give me as much experimental info as possible. best, Christian Quote Link to comment Share on other sites More sharing options...
Guest Rain Posted April 17 Report Share Posted April 17 On 4/14/2023 at 7:33 PM, Christian@mabtech.com said: The spots look rather large and blurry, yes. Is this a precoated kit you purchased from Mabtech or have you coated the plates yourself? What concentration of capture mab did you use? Are these mouse splenocytes incubated for a long time in the plate? Please give me as much experimental info as possible. best, Christian The kit was coated by myself, and the concentration of capture antibody was 15ug/ml. These are mouse splenocytes, we incubated for 36h. We fogot to detach the underdrain before drying. Could this matter? Quote Link to comment Share on other sites More sharing options...
Christian@mabtech.com Posted April 17 Report Share Posted April 17 No the underdrain has no impact on this phenomenon I would say. What substrate did you use? Mabtech? When you coated the plate, did you use etoh pre-activation of the PVDF membrane? Quote Link to comment Share on other sites More sharing options...
Guest Rain Posted April 17 Report Share Posted April 17 7 hours ago, Christian@mabtech.com said: No the underdrain has no impact on this phenomenon I would say. What substrate did you use? Mabtech? When you coated the plate, did you use etoh pre-activation of the PVDF membrane? Yes, I used Mabtech substrate for ELISpot. And I used etoh pre-activation of the membrane. I forgot to filtrate the substrate with 0.45um membrane. Quote Link to comment Share on other sites More sharing options...
Christian@mabtech.com Posted July 20 Report Share Posted July 20 Dear Rain, I am sorry for not getting back to you in this thread. The notification system is not great. I would very much recommend you reach out to Mabtech and get a precoated mouse IFNg plate for testing. Exclude the possiblity of something going wrong in the coating process. Sometimes the etoh activation process can be tricky. Another alternative is that your mice splenocytes have very poor viability and that 36h is too long. Hence I would reach out to a Mabtech distributor or Mabtech HQ and ask for a precoated mouse plate. Re-run the experiment with your own plate in parallell and observe the difference. Also, in order to exclude the possibility of really poor cell quality, see you can get a hold of freshly isolated mouse splenocytes. Quote Link to comment Share on other sites More sharing options...
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