Spots in no stimuli wells when using mouse IFNg splenocytes

[Guest Subeena Sood]

Dear Technical support, 

we had been using Mabtech ELISPOT plate 3321-4AST for about a year now. We recently (beginning of this year) started to observe high background in our no stimuli well. The example plate is attached as a ppt. We have troubleshooted almost everything, but still high background. This plate is showing background in group D, but it sometimes shows up in PBS control animals or just random groups, nothing to pin point to the treatments that these animals are getting. We use CTL media supplemented with 1% Glutamine and penn strep for spleen cell isolation. We always use fresh spleen cells. Here is our spleen isolation steps and we use MACS quanta dissociator: 

Dissociate spleens on MACS Quant

1. Collect spleens from mice and keep on ice. *Set centrifuge to 4C*

2. Close tube lids quarter turn past stop and put on MACS Quant dissociator.

3. Select tubes on the screen and select program for mouse spleen cells (~60sec)

4. Start program. Tap tubes on screen and press ok after program ends.

5. Rerun program multiple times (at least 2-3 times or as needed). Check there is no spleen stuck in the blades of the tube

 

V. Filter splenocytes (70um filter)

1. Place strainer over 50mL tube and prewet with 3mL media

2. Pour spleen slurry from C tube over filter

3. Rinse tube 2x with media (wash with 3mL then 2mL)

4. Centrifuge cells 10 min at 4C at 300g

5. discard supernatant and resuspend the pellet in 15ml of CTL media. 

6. Our usual cell counts is between 15-25million cells/ml. 

We have also tried manual technique of meshing the spleens by the using the back of the syringe, but still we see spots on negative stimuli wells randomly. There is no correlation between the cell viability or high background. if you want we can even send you another ppt from manual vs MACS dissociator. and moreover this problem started more when we ordered the new batch of kit from Mabtech in Nov.  

Is there a possibility of leak happening behind the membrane i.e., PMA is leaking into the wells above? Any suggestions or ideas will be helpful. 

Thanks

subeena sood

mabtech-troubleshoot.pptx

[Guest Subeena]

Sorry, forgot to add the title to this query. 

"Spots in no stimuli wells"

[Christian@mabtech.com]

Dear Subeena, welcome to the Mabtech forum!

In your PPT I can see that your spots are of excellent quality but that you do indeed suffer from higher backgrounds, especially in group D. 

Here are 3 comments/questions:

1. At Mabtech we are familiar with transporting spleens on ice, but all our prepping is done at room temperature. How long are the cells kept at 4C in stage 1 of your MACS quant? This might not be the reason for your backgrounds but I want to highlight that stressed cells are never good in ELISpot. For flow it does not matter. 

2. CTL media is reported as causing higher IFNg backgrounds in ELISpot. I would instead recommend using RPMI1640 supplemented with 10% FBS of excellent quality. Media can make a huge difference to backgrounds. If you have to use a serum free media we tend to recommend AIM-V. 

3. The high background could reflect the in vivo activation of T-cells in the donor mouse. Have they been treated in some special way? Sometimes projects expose the mice to a virus vector. The control group is the vector but without the encoded protein. However, the vector on itself causes immune activation and will generate backgrounds in mouse IFNg. It is always good to have control mice that have only been injected with PBS as a "true" negative control. 

Medium ELISpot 2010.pdf

[Guest Subeena]

Dear Christian, 

Thanks for your reply and your comments. Just few more clarifications regarding your suggestions. 

1. So you are suggesting we collect on ice, but then after homogenization do everything at RT. We keep the cells on ice, including centrifugation at 4C till the time we make working conc. for plating the cells at 250,000 cells/well. 

2. Do you think we should block the plate with media supplemented with 10%FBS to reduce background?

3. We always have PBS control mice in our studies, but sometimes (rarely) we do see spots in that group too. 

4. Do you think we should reduce our incubation times from ~21h - 18h? 

5. substrate development step is done for 15min. But we can reduce that? 

6. So there is no possibility of leaking PMA from the back side of the plate? 

Thanks

Subeena

[Christian@mabtech.com]

Hey,

1. If you quickly get the spleens in your lab after the mice are sacrificed I would only work at room temperature. If there is long transport in car from where the mice are sacrificed to your laboratory, we also keep the spleens on ice during the transportation phase. However, we immediatly start working at room temp once spleens arrive in our hands. If you have direct access, work at room temp. This is unlikely to be the source of your problem but I am sure it is better. 

2. Normally serum free media should contain plenty of protein that will block the membrane effeciently. But I am not familiar with CTL media. You can certainly try seeing if blocking with 10% FBS improves the situation. All ELISpot should always be performed with blocked membranes. It is part of the protocol. 

3. Ok so when you run PBS control mice you get almost zero spots in unstimulated controls? Do I understand correct? If yes then the backgrounds obviously originates from in vivo activated T-cells or NK cells. Then there is nothing wrong with your experimental setup. Please confirm what you meant.

4. Will make no difference. 

5. Sure you can reduce it, but the background observed is signficant. These are strong spots and they wont go away when lowering to 10min development time. 

6. There is no possability of leakage between wells. However, when you set up the experiment, during the seeding of cells into the ELISpot plate, it is easy to make a mistake and contaminate the unstimulated wells. You always have to change pipette tips for every stimuli category. If you take cells from one donor, and into antigen wells, then go back into the container with cells again and draw again, small droplets of antigen can be stuck on the outside of the pipette which will then contaminate the "motherstock". Game over.