Blank areas in IFNg ELISpot with human PBMCs

[Guest Lena]

Hello! 

I have been conducting IFNg ELISpot assays with human PBMCs for a while now, but recently I am observing blank areas in my wells (either no spots at all or much less than in the rest of the well) - it can be seen in most of the wells of the attached picture. I tried changing the ethanol-treatment of the membrane, putting the cells through a cell strainer before plating etc., but nothing seems to help. I don't really know what else to do, especially as the spot distribution was great 2 months ago using the same protocol. 

Would could cause this and how can I solve this issue? 

Thank you very much in advance, 

Lena 

[Jens@mabtech.com]

Hej Lena!

It looks like you are experiencing the exact same issue as has been discussed in this thread: 

Briefly, probably some reagent have not reached the bottom of the well. We have seen these blank wells a couple of times, and we have yet to experience a blank well that cannot be "rescued" by repeating the detection steps again. Thus, the problem is not a lack of coated capture antibodies, but a one failed detection step. Please repeat the detection steps in the blank wells as follows: 

1. Wash the blank wells 5x with 200ul PBS.

2. Add detection antibody at 1ug/ml in PBS containing 0.5% FCS and incubate for 2h.

3. Wash the blank wells 5x with 200ul PBS.

4. Add SA-ALP in PBS containing 0.5% FCS and incubate for 1h.

5. Wash the blank wells 5x with 200ul PBS.

6. Add substrate and incubate for 10min.

I would be surprised if you didn't get results looking like this: 

 

So what to do to avoid blank wells in future experiments? Well, if you are too careful with emptying the pipette, there is a risk for a bubble formation in the bottom of the well, that blocks the following reagent to reach the bottom. Solution:

•Look carefully at the plate for odd looking wells after addition of liquid.

•Remove bubbles by tapping the plate against the bench.

•Do not touch inner wall of the well with the pipette tip.

 

Hope you manage to resolve the issue! Please come back to us if you don't (or, yeah, please do if you resolve it as well, would be nice for us to know :))

[Guest Lena]

Hello Jens, 

Sorry, maybe I should have been clearer, but I am not concerned by completely empty wells, but by the "pattern" that is seen in some wells. So some areas in the certain wells that show no spots or very few and the cells seem to be pushed outside. I wash the plate with a multipipette and I am rather careful, so I don't think it is caused by too aggressive washing. 

Thank you, 

Lena 

[Jens@mabtech.com]

Oh, I see! It looks like the cells have been "pushed" to the side of the well. That might happen if you add the cell suspension first and the stimulus after. In what order are you adding reagents?

The best way is to first add medium including (or not including) the stimuli and the cell suspension last. Use for example 50% of the final medium volume for the stimuli (or just medium for negative control) and 50% of the final volume for the cell suspension.

 

 

 

[Guest Lena]

Indeed I plate the cells first - I add 100 µL cells and then 10 µL of the stimulus. I will try adding the stimulus in 50 µL medium first. 

Thank you very much for the quick support!