Purple spot background

[Guest Idania]

Hi Christian,

We are experience a problem with purple haze background in some wells. We are using the IFNg coating plates from Mabtech, so no need for EtOH activation of the membrane. We are testing human PBMCs response against peptides. Initially, we were washing with PBS as recommended, but, because now we are doing ELISpot after long term culture of the cells, we were having high background in the negative control. Thus, to try to decrease the background, we added 5 wash with PBS-0.05% Tween after cell stimulation following by 5 wash with PBS and we continue using only PBS for the other washes. Most of the plates are developing well without the purple color; but sometime, some wells do not develop properly and get the purple haze that interfere with the spot development and counting. Please see below the picture of one of the last plates (please check row D), the purple haze happens only in some wells and not in all the plate. 

I really appreciate if you can give us some feedback why is this happen in some wells, how can we solve this problem? Or any suggestion that you have to reduce background when using long term cultured PBMC without using PBS-Tween.

Thank you for your help,

                                     Idania

 

IFNg-ELISpot plate 1.tif

[Christian@mabtech.com]

Dear Idania,

I would say in general that you results look very good overall. There would be no difficulty counting this plate accuratly on IRIS or ASTOR. But I see you are using another reader where a tone difference in membrane-color throws off the spot-counting algorithm. I understand the problem. 

Well D4-D9 are confluent with spots and the substrate often leave a slight purple tone to them. However, the membrane color difference between well D1 and D2 is striking. 

I think the use of tween can contribute to the problem you are seeing. I therefore would suggest another protocol for getting rid of cells stuck to membrane:

1. After incubation of the plate is over, wash with pure PBS 5x200ul. 

2. After this add 100ul of 1mM EDTA diluted in PBS to all wells. Place the plate in the incubator for 10min. The EDTA in combination with heat with will make cells dislodge from membrane.

3. Wash in pure PBS 5x200ul.

4. Add detection antibody and continue protocol as normal. 

5. When turning off the substrate reaction use pure PBS. Not tap water. 

 

Try this and see if it improves the purple hays.