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'Cells alone'/'no stimuli' wells with high number of spots

Guest Carlos2022-08-10T17:33:56Z

Hi there!

I have issues with my mouse IFNg ELISpots similar to this post:

From reading previous troubleshooting conversations, I can see that a probable reason could be the in vivo activation of T cells... And if this is the case, what is the solution?

My latest ELISpot had crazy spot numbers for my 'cells alone' wells, for example 617 and 477 for one treatment group, 339 and 161 for another group. From looking at the data, it seems like each mouse has a similar trend of overall spot count. For example, the mouse with 617 spots in the 'cells alone' wells (average triplicate), had the following spot counts in the 7 different peptide wells: 600, 800 (TNTC), 800 (TNTC), 657, 654, 619, 577; the mouse with 477 spots in the 'cells alone' wells had the following spot counts in the 7 peptide wells: 529, 800 (TNTC), 800 (TNTC), 541, 583, 499, 549; and as a last example, the mouse with 161 spots in the 'cells alone' wells had the following spot counts: 154, 200, 202, 179, 206, 181, 159. As you can see, there is a trend in each mouse, which I assume could be due to in vivo T cell activation as previously mentioned? Just to different extents...?

Previous ELISpot experiments have shown lower spot counts overall, so I'm not sure what could have been done differently this time... (I always make sure I pipete the 'cells alone' wells very carefully, trying to isolate these wells so to not mix them with peptide-containing wells, and I do the same with the positive control wells (concanavalin A). I suppose I will have to see what my results are like in my next ELISpot, but I just thought I'd ask in case there is some sort of explanation. I am particularly curious as to what can be done if the T cells are indeed activated in vivo? Does this simply mean that the wells will all have many spots and, if so, what can be done about this? Would lowering splenocytes/well help? (We use 500,000 splenocytes/well).

Thank you in advance for your help!

Guest Carlos2022-08-17T18:44:56Z

@Christian@mabtech.com could you please help me?
Kind regards,
Carlos.

Christian@mabtech.com2022-08-17T21:07:00Z

Admin

Sorry Carlos!

High backgrounds in mouse splenocytes can ofcourse have a myriad of reasons why. Can you tell me more about the experimental setup? These mice have been injected with something and you take the spleen how long after last injection?

Sometimes people use adeno-vectors for injecting a vaccine candidate in their mice. The ctrl they use only get the adeno-vector but without the sequences for the vaccine. They then have very high backgrounds and find this strange. However, the adenovector itself is immune-stimulatory. The mice become in vivo activated and T-cells and/or NK cells secrete a lot of IFNg in the spleen which then show up in the ELISpot. Thus, it is good to have a control mice in your experiment that have only been injected with PBS, nothing else.

Are you isolating the splenocytes and runnning the ELISpot directly, or do you freeze the cells and run ELISpot at a later timepoint? How does you protocol for isolating splenocytes look? How is you freeze and thawing protocol?

When running ELISpot you can get weird results if the FBS used in the cell culture media is not good. What is you cell media?

Please share some images if you can. Add high resolution images into PPT and upload. Explain in textboxes what is what. Number of cells/well and stimuli. Include images of unstim and antigen wells. 500K cells/well is a bit high but nothing outrageous. We usually recommend 250K cells/well.

Many times I come across people that do have IFNg backgrounds but they also have clear antigen specific response as indicated by much bigger spots in the antigen wells. However, do to allowing the substrate to develop the spots for too long the antigen wells get almost blacked out whereas unstimulated wells still get counted rather accurately by the ELISpot reader. You loose stimulation index. In this scenario it can be better to lower cell counts and lower the substrate incubation time. Then optimize count settings on your reader to "capture" the true response that visible to ones eyes. Share some images!

Guest Carlos2022-08-18T13:00:26Z

Dear Christian,
I have attached the edited image as you requested. I hope you can see it well enough.
To answer your questions:

The spleens are harvested one week after the last injection.
The splenocytes are isolated and used directly for the ELISpot, without freezing/storing. We extract them by flushing them with T cell medium (recipe below) using needle and syringe, followed by sieving with cell strainer and centrifugation for counting prior to plating.
T cell medium recipe is RPMI with 10% FBS, 5mL 200mM L-glutamine, 10mL 1M HEPES, 5mL 100X pen/strep, 500uL Fungizone (supplements sterile-filtered).

The experimental design was as follows:

Group 1 (mice 1 and 2 - mouse 3 was culled and excluded): received peptides 2 and 3 on Days 1, 15 and 29; and peptide 1 on Days 2, 16 and 30.
Group 2 (mice 4 and 5 - mouse 6 was culled and excluded): received peptides 1, 2 and 3 on Days 1, 15 and 29.
Group 3 (mice 7, 8 and 9): received two DNA bullets encoding for the proteins corresponding to peptides 2 and 3.

The images explained:

Red is positive control (Concanavalin A).
Blue is cells alone/no stimulation.
Yellow is peptide 1 (each row is a different, shorter peptide derived from peptide 1).
Green is peptide 2 (each row is a different, shorter peptide derived from peptide 2).
Orange is peptide 3 (each row is a different, shorter peptide derived from peptide 3).

Apologies for the confusing plate layouts. As you can see, most wells have many spots, including blue squares (unstimulated cells). We know from previous experiments that peptide 1 (yellow) is very immunogenic, hence the higher amount of spots compared to the other peptides. Also, positive controls (red) are too numerous to count.

Thank you so much for your help. Kind regards!

Christian@mabtech.com2022-08-29T12:06:53Z

Admin

Again, I am sorry for the late reply. For some reason I dont get a proper notification when unregistered users update a forum thread.

Looking at your data it certainly looks like you have in vivo activated T-cells.

1 week post last injection could be too early. It is better to take the splenocytes 2-3 weeks after last injection in order to pre-activation.

Finally, good to include mice you only inject with PBS as a control.

Guest Lauren Cave2024-08-02T15:00:19Z

Hi,

I was just reading this conversation as I too have spots in my PBS treated, unstimulated ELISPOT samples and I was just curious because I did my ELISPOT with frozen mouse splenocyte samples - can there be non-specific spots formed because they were frozen instead of fresh samples?

Kind regards,

Lauren

admin2024-08-03T11:26:31Z

Admin

20 hours ago, Guest Lauren Cave said:

Hi,

I was just reading this conversation as I too have spots in my PBS treated, unstimulated ELISPOT samples and I was just curious because I did my ELISPOT with frozen mouse splenocyte samples - can there be non-specific spots formed because they were frozen instead of fresh samples?

Kind regards,

Lauren

IFNg secretion from unstimulated mouse splenocytes can be caused by many factors. When you work with frozen splenocytes there are added factors involved related to the freezing and thawing protocols.

Things to consider:

1. The immune system of the mice could be activated affecting the isolated cells. Unlikely with PBS treated control mice.

2. FICOL isolation could be done incorrectly which increases IFNg backgrounds. Freezing protocol can also be done incorrectly leading to T-cell activation.

3. The FBS used is full of contaminants activating the T-cells.

4. Thawing protocol of frozen pbmc is done incorrect causing Tcell activation.

5. Contaminated cell culture media.

To name a few.....