Increasing spot presence in negative controls

[Guest Renee]

We have recently switched to using your pre-coated IFN-g Elispot plates (3420-4AST plates and strips) and have noticed an unusual phenomena that I'm hoping you can help me out with.

 

IMAGE ATTACHED BELOW

We are running human PBMCs in triplicate in a serial dilution. A1-3 contains the positive control, B1-3 is the negative control and C1-3 contains our test antigen. This pattern repeats for the remainder of the plate below. (D1-3: Positive, E1-3: Negative, F1-3: test etc.) Row 1 contains 100 000 cells, row 2 - 50 000 cells and row 3 has 25 000 cells. This dilution is done with a multi-channel pipette. We have noticed that instead of the spot number decreasing as expected (and as in seen in the positive and test conditions) it appears to increase as the number of cells decrease in the negative control. This has been observed consistently over about 100 cell samples now. Any ideas what might be causing this?

[Christian@mabtech.com]

Dear Renee, welcome to the forum!

 

This is an interesting case and I cannot immediatly come up with a reasonble explanation but I do have some initial comments/questions:

1. Can you attach well images of higher resolution? The attached plate JPEG is not the best resolution so it becomes tricky to see details. In particular I would want to see well E1, E2 and E3 plus C1, C2 and C3.

2. What can you tell me about your PBMCs? Are they freshly isolated cells that have immediatly been added to the ELISpot plate? Or have they been cultured before hand in some special way?

3. One problem I see is that you AID reader is not optimally configured in terms of count settings. Take wells C1, C2 and C3 as an example. I can visually see that well C1 contains many more spots than well C3, but the spot-count show the opposite. This problem is probably also affecting you negative controls but I can see examples were the effect you are talking about seems to be for real. Anyway, the count settings of your machine should be adjusted. Please have a look at this Youtube video:

 

 

 

[Guest Renee]

Thank you for your speedy reply. I have started to watch the tutorial but I am obviously working off an older version (Version 6.0) as "enzymatic optimised" is not even an option in the camera initialisation parameters. We currently have ours set on "full resolution". The machine has not been optimised for our purpose and I have been struggling to obtain clean images of the wells despite altering the camera settings. The spots always appear slightly blurred. I have been manually adding and subtracting spots which has been time consuming. 

Do you think upgrading the software would help?

The cells we are running have been isolated from human blood and immediately stored at -80C. Cells are thawed, treated with DNase and rested for an hour prior to addition to the plate. We do not use any serum during this process. 

 

It seems the reading on C1 was a glich. I have altered the intensity settings and the count has increased to 526.

Please see the requested wells attached below.

C1

C2

C3

E1

 

E2

 

E3

 

 

 

 

[Guest Renee]

Apologies for the multiple pictures. The first three are wells C1, C2 and C3 and the last three are E1, E2 and E3

[Guest Renee]

Just a side note, the images of the wells appear clearer in the message above than they do on the analysis software. Would this be a version issue also?

[Christian@mabtech.com]

Hey Renee,

Thank you for the upload of photos! The ELISpot look really great! Its good to use pre-coated plates

The images also have led me to conclude a few things:

1. My guess is that this plate has not been developed with substrate for an extensive period of time, probably around 10 minutes or even lower. Substrate developement is naturally a big part of how strong spots become but it also leads to higher backgrounds the longer you develop. In this case, I think you have done an excellent job with substrate development. Just right if you ask me. Antigen stimulated spots are easy to read once the reader is properly configured. However, from the perspective of the unstimulated controls, substrate developement was just slighly too short. You can visually see that there are more spots in E1 compared to E3 if you look closely, but due to short substrate development time, the tiny spots in E1 has not been given its full "darkness" and development. In the unstimulated E3 there are fewer spots and the with the time for substrate development time given, they have had more oppertunity to develop stronger. In E1 with many more spots, the time that was given with substrate limited their development. Had you developed for longer, I believe that unstimulated wells would have better reflected your titration of cells. At the same time, you would have risked overdeveloping your antigen stimulated wells with confluent spots as a consequence. In summary, I think you have done it correctly and this can be compensated with different count settings. 

2.  With a better configured reader I think the differences you show in your initital post could be greatly reduced. Even tough you are on an old software, the same principles of the Youtube clip can sort of be applied. Instead of algorithm C that I recommend on youtube, try algorithm B with settings of 10:10:10. 

3. When it comes to the clarity of images inside of the AID software. I think this could be related to a setting called anti-aliasing. It should be checked in the software. I think I mention it in the youtube video but only briefly. Otherwise, check my attachment were I have highlighted the setting in my version. Even tough you are on a older software, it should be in a similar place.

Anti-aliasing in AID readers.pptx

[Guest Renee]

Thank you so much. I've only just had a chance to implement these ideas and they have made such a difference!

[Christian@mabtech.com]

No problem Renee, glad we could help!