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too many negative spot of IFNg ELISpot


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I assayed and the negative spots are too many to accept the result of samples by criteria( < 12) . I cultured 2E5 cells/well with/without stimulus and I assayed IFNg ELISpot(ALP). 

I stopped the color development as repeating to add and remove tap water, however, the spots did not stop immediately and they kept emerging while I stopped the wells developments with 8 multichannel pipette.  Is there any way to stop at once like ELISA or skill to stop the color development?  


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Stopping the substrate reaction with water or PBS will terminate the development of spots immediately. However, deciding on a time of when to do this can be tricky. Many publications of ELISpot say that "spots were developed until distinct spots were seen on the membrane". The problem with this approach is that it becomes very subjective, one user might go for 10 minutes, another for 25 minutes. This will have a huge impact on the result. 

My recommendation is to pick a time for substrate development and stick to it. For ALP I often like somewhere around 10minutes. The problem with judging the development time by eye is that the substrate forms a meniscus which leads to spots looking smaller than they actually are. Also, when plates are fully dried faint spots will be much more apparent compared to when the membrane is wet. 

Looking at your images it seems to me that you have stopped the development rather quickly, which I think is good. You have not overdeveloped. Instead, you have high IFNg backgrounds in your cell samples. It is a "true" result, not an artifact.

High backgrounds in IFNg ELISpot can have many reasons but in general these are the main aspects:

- Poor viability and alot of apopotic cells can lead to higher backgrounds. Viable cells get affected by the products produced by the dying cells. 

- Cells come from donor that have in vivo activated T-cells due to inflammation, cancer, autoimmune disease or perhaps stress.

- Cells could be totally fine but inappropriate cell handling during isolation has made them irritated causing backgrounds. Cell handling involves the entire chain, from that blood is retrieved through venipuncture to having a cell suspension being seeded in the ELISpot plate. If you are running frozen samples of PBMC, the freezing and thawing protocol can have huge impact on IFNg backgrounds. 

- Fetal Bovine Serum used in the cell culture medium comes from a bad batch. This can cause high backgrounds in IFNg ELISpot.

- During setup of the assay the cell culture is contaminated with something. 

These are the main aspects I would say. 

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