High Background in IFNg ELISpot for mouse spleenocytes

[Guest Sajib]

Hi,

I am also getting high background for  Mouse IFN-gamma ELISPOT assay from fresh  Mouse spelenocytes. Initially, I inject AAV in the animal via intraperitoneal administration and after 9 days I conduct in vitro stimulation with my peptides in ELISpot plate (MSIPS4W10, sigma). I used  300000 cells per wells. I used ConA as positive control (2ug/ml). Stimulation time was 24 hours in suitable condition. As culture media, I use RPMI with 10% FBS as well as CTL media ( serum free media, Immunopot. After that I follow all the steps stated on the protocol from MABTECH. I used BCIP-NBT substrate with 5 minutes incubation and then wash with water . But The back ground was still there. Please see the attached ppt for images.

IFNg Background.pptx

[Christian@mabtech.com]

Hi Sajib,

Looking at your PPT presentation it is clear to me that these are correct real spots. They are not artefacts, ie they represent true IFNg cytokine secretion. It either comes from something in cell handling or is a true representation of what is going on in vivo in these mice. The positive control ConA in well A4 induces stronger IFNg responses. 

What is the immunological background of these mice? Something in their biology that could explain a high level of IFNg release in their spleens?  

How do you isolate the splenocytes? 

We know from experience that CTL media induces unwanted backgrounds in IFNg. They have versions of their media that contains factors that induces IFNg secretion. Did the result look the same when you used RPMI1640 with good quality FBS? 

[Guest Sajib]

Hi 

Thank you so much for your response.

Would you please let  me know what do you mean by immunological background?

• Spleen isolation was conducted as follows:
•  Put spleen in cell strainer
•  Put 5ml of RPMI+10% FBS media (warmed to 37^oC)
• Use the syringe plunger to gently but firmly break down the membrane of the sple
• Spin the cells in the tubes at 1250rpm (350g) for 5 min
• Aspirate supernatant and vortex the cell pallet to loosen the cells
• Add 500 ul of ACK lysis buffer for 1 spleen for 40 sec
•  Stop the lysis by adding 10x more media.
•  Spin and Resuspend in 5 ml of warm media (RPMI media +10% FBS) (warmed to 37^oC) and count

We observed the similar backgound for IFNg when used RPMI+10% FBS and thus started using CTL test media assuming that FBS is responsible for providing the background spots. I forgot to mention that we use Biostack machine to coat, wash and block the plates.

[Christian@mabtech.com]

The protocol for isolating your splenocytes look fine.

Since you are injecting an AAV vector into your mice, it is possible that the vector persist in the cells for long periods of time post injection? Reading a bit on wikipedia about AAV:

"AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state"

Thus it is not far fetched that TLR7/8 will be stimulated for long periods of time post injection causing IFNg release in NK cells for example. 

It would be good if you had control mice in your experimental setup were you inject only PBS (and not AAV). These mice can then serve as a control for your AAV. Doable?

 

Another idea: Possible that the Biostack machine contain contaminating agents in their tubing or something like that? If the capture antibody gets contaminated with TLR ligands for example this can induce high levels of backgrounds in ELISpot. 

 

With immunological background I was simply asking if the mice perphaps came from a very special mouse model? Say an inflammatory mice model for example.

 

[Guest Sajib]

Hi 

Thank you so much for the response. We observed similar response from Control mice ( injected with PBS, see the attached ppt. Our ELISpot results for IL2 works very fine with little and no background as well as desired response for positive peptides. These are traditional BALB/C mice.

[Christian@mabtech.com]

Having contemplated this over the weekend I realized something:

- IFNg is secreted by T-cells but also by NK cells and innate lymphoid cells. 

- By contrast, IL-2 is more T-cell restricted. I have not heard of NK cells or innate immunity cells being able to secrete IL-2

Could it be that you are obeserving an NK cells derived IFNg response as a result of the AAV? That is my best guess. 
 

Would it be possible for you to use IL-2 in your study? Mabtech has a great IL-2 system:

https://www.mabtech.com/products/mouse-il-2-elispot-basic-kit-kit-alp_3441-2a

 

I never saw the attached PPT with your injected PBS results. Would be interesting to see how it looks!