About precipitate, membrane staining and negative controls in ELISpot assay

[Yaice Sandoval]

Hi, dear support team of MABTECH

Our team are working on an Elispot IFN-g with chicken splenocytes using the MABTECH kit  ELISpot PLUS: Chicken IFN-g (ALP). We have been reading this forum in order to learn from your advice.  Now we have some specific doubts that we would like to clarify.

-We are following the exact instructions of the kit and in the past experiments, we had really nice results for the standardization of the conditions (Standarizationp1PHA7.jpg). In our last experiment (Lectura07no2022.jpg), we saw two things that we hadn't seen before. In almost all our wells we have kind of a precipitate. It can be seen clearly in row B, where we have no stimuli to the cells. Additionally, in some of our groups, we have membrane staining in some wells of columns 6-8. Could you give us any advice or do you have any idea of what could be happening?

-Related to the spot number per well: Do you recommend a specific range of spot number per well? We have prove different cell concentrations (from 100,000-500,000 cells/well) and we selected 300,000 cells/well because we had <500 spots. Recently, we prove perfusion as a new method to set apart the splenocytes. We got ~99% of viability, so now we have much more spots than initially with cell grinder (80-90% viability).

-Finally, we would like  to ask about the negative controls. We had been using a synthetic peptide with a random sequence that in appearance is not related to our antigen (in fact it have 0 % identity in sequence) but at the ELISPOT assay we are detecting that it produce almost the same reaction than our synthetic peptides related to our antigens (100 % identity to the antigen). Do you have any comment for these or could you give us a recommendation for negative control?

 

 

Thank you in advance!

Regards

[Christian@mabtech.com]

In your past experiment, 1PHA7, the spots look fantastic. Unstimulted controls (I presume) are on the left hand side and antigen stimulated wells on the right. Exactly what you want to see in a successful ELISpot experiment. 

Your last experiment It looks to me like you have very high backgrounds. Is the layout of this plate the same as the previous one? with unstimulated controls on the left hand side? For some reason, the chicken cells you are evaluating are secreting IFNg even in the absence of stimuli. To me, it looks like they are in vivo activated. Any special reason as to why this particular donor chicken would have in vivo activated cells but not in the first experiment? 

How does the injection protocol look for the chickens? If splenocytes are taken too early after the last injection you can get something called hyperimmunization where the T-cells are in vivo activated. 

When you say "precipitate" I dont fully understand. To me it looks like you have massive number of spots/well. Each spot is made up of a "precipiate" from the ELISpot substrate, so that is normal. With massive number of spots they can get this "pink" tone to them during development. Different protocols for shutting off the substrate reaction can lead to different tones in color of the spots. 

99% viability sounds a lot better than 80-90%. But is it so that your last experiment you got after using perfusion whereas the first was achieved with grinder?

By the way, I will keep this thread going but delete the other one where you posted as guest. Welcome to the forum!

 

[Yaice Sandoval]

Hi Christian! Thank you so much for your answer. 

On 11/8/2022 at 7:58 AM, Christian@mabtech.com said:

I will keep this thread going but delete the other one where you posted as guest.

 Yes, thank you. I read in another post that you have troubles  with the notifications when someone make an entry as a guest, so I re-sent my post. 

I prepared some slides in .pptx to show some images and explain a little more in the notes box. The short versions, is that the plates have different layout.

With this "precipitate" we are not sure if in the non-stimulated cells we have a very high background. But, where we are seen a lot of spots (and we should not been)  is in our negative control (non-related peptide). We are searching any explanation for these. We are not sure if I we could be seen an in vivo activated cells response.

On 11/8/2022 at 7:58 AM, Christian@mabtech.com said:

If splenocytes are taken too early after the last injection you can get something called hyperimmunization where the T-cells are in vivo activated. 

Do you recommend any specific time?  The cells we are using in this assays have 15 days post-immunization.

 

Thank you in advance.

Regards.

Mabtech_forum_YaiceSandoval.pptx

[Christian@mabtech.com]

Hi again Yaice,

Thank you for the explaining PPT. I now understand your experiments better. 

The massive background observed in your second experiment where the membrane is filled with weird looking "spot-like" structures looks very strange. Could be artefacts but I am not 100% sure. When you isolated the splenocytes from this particular chicken was there anything that stood out? Was the spleen extra large compared to your other experiments? Did you get a lot of contaminating red blood cells in your splenocytes? Maybe you could share your protocol for isolating splenocytes? Do you run freshly isolated cells or do you freeze them and run thawed samples in the ELISpot?

In your explaining PPT you also show "non-vaccinated" samples still having very high backgrounds. Just for clarification: Are these splenocytes from Chickens only injected with PBS where you have taken the spleen and isolated splenocytes and finally run the samples immediately in ELISpot?   

In the first example you shared where wells look clean and nice (no artefacts) and spots are normal I would say that backgrounds are rather high in the medium control. In addition, you have stimulated with "non-related peptide", does this mean that you expect NOT to see any response? There does not seem to be increase in IFNg secretion in these wells. The PHA response is rather weak but that is in line with our own observations. We recommend PMA+ionomycin instead.     

 

I looked back in our R&D data found a splenocyte evaluation we had done. Here we used 1 million cells/well. Much higher than when working with human cells. With no stimuli we sometime got backgrounds in some chickens but it is lower compared to what you are seeing. I am therefore interested to know more about your isolation protocol and if you are running fresh or frozen samples. 

Furthermore, we used PMA+inomycin for positive control as that worked much better than PHA. Here we recommend not using 1 million cells per well with PMA+ionomycin but 500K/well might be good. 

 

[Yaice Sandoval]

 

Hi, Christian!

When you isolated the splenocytes from this particular chicken was there anything that stood out?  We didn’t notice something different in the spleens. The difference here is the separation method between grinder (1st experiment) vs perfusion (2nd experiment).

Do you run freshly isolated cells or do you freeze them and run thawed samples in the ELISpot?  We run the experiment with freshly isolated cells. In the past, we intended to freeze cells in order to have a backup but we didn’t get good results (Again, we read that freezing is not recommended).

Here is our protocol to isolate:

-Chickens are sacrificed and the spleens we preserved in DPBS-Pen/Strep at 4 ºC (we preserve it on ice only the time they are transported to the lab). We read in another post that you recommend manipulating the cells at room temperature, so we implement that.
-We grind/perfuse and collect the cells. To isolate cells we use an Histopaque 1077 gradient.
-We recover the mononuclear cells layer. Then we wash two cycles with DPBS and then we continue with the MABTECH protocol.

Are these splenocytes from Chickens only injected with PBS where you have taken the spleen and isolated splenocytes and finally run the samples immediately in ELISpot? They are not injected with PBS. In fact, these spleens come from recent hatch-out chickens (~1 day).  We run the ELISpot immediately.

About to the “spot-like”, do you think that could be an artifact related to the antibodies? For other protocols, we filtrate the antibodies solutions.

Finally, thank you for sharing images and information about your experiments. What do you use as negative control? In some papers we have seen that DMSO is used as control.

 

 

Thank you in advance

[Christian@mabtech.com]

Ok, I would recommend grinder then for future experiments. I am not familiar with perfusion but it might be the reason for your very strange backgrounds. 

When it comes to your protocol I have one comment

Do you really need to use Histopaque 1077 gradient? We don't. Here is our protocol:

Steps

Pour out the spleen into a petri dish. If required, remove access fat and tissue using scissors and tweezers or forceps.

Place a cell strainer in a new petri dish and transfer the spleen to the strainer. Add 5 ml medium.

Use the flat plunger end of a syringe to homogenize the spleen through the cell strainer into the petri dish. 

Rinse the cell strainer with 5 ml medium into the petri dish. 

Transfer the splenocytes from the petri dish to a 15 ml centrifuge tube. 

Leave the tube for one minute to let big clumps sediment or remove clumps and aggregates using a sterile Pasteur pipette. Transfer the splenocyte solution to a new 15 ml centrifuge tube. Fill the tube with medium.

Centrifuge for 10 minutes at 300 x g. 

Discard the supernatant and resuspend the cell pellet in 15 ml medium.

Note the volume and take a small aliquot to count the cells. 

Centrifuge for 10 minutes at 300 x g.
Tip! Count the cells during this step.

If you want to use the cells right away 
Discard the supernatant and resuspend the cell pellet in appropriate cell culture medium by gently pipetting up and down. The splenocytes are now ready to be used for cell-based immunoassays such as ELISpot, FluoroSpot and flow cytometry.

 

I think this can improve your results. 

Furthermore, incubate your splenocytes at 1 million cells/well. High number but required for detecting antigen responses as seen from the experiment I shared. For unstimulated control we only incubate the splenocytes in cell culture media. Nothing else.