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No clear spots in mouse IFN-γ ELISpot


Guest Rain
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Guest Rain

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

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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

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Guest Rain
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?

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Guest Rain
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.

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