Purple center area

[Jun]

Dear Mabtech staff and researchers,

 

I am conducting MHV(murine hepatitis virus) Surface protein peptide-specific T cell ELISPOT assay.

using Elispot plate: M8IPS4510 | MultiScreenHTS IP Filter Plate, 0.45 µm, clear, non-sterile

Primary IFN-g antibody (capture): BD cat no. 554430, final concentration in Elispot plate is 3.0 µg/ml 

Secondary IFN-g antibody (biotinylated; for detection): BD cat no. 554410, final concentration in Elispot plate is 2.0 µg/ml 

Positive control: PHA-M (SIGMA L8902-5MG), final concentration in Elispot plate:  2 µg/ml

I seeded 1,000,000 cells/well splenocytes from MHV infected mice.

Attached is the image of the result.

 

As we can see the images, the center of some wells have purple area. Have you ever had such experiences?

I thought it was because of ethanol when pre coating. Do you think is it meaningful without ethanol ELISPOT?

Finally, we could not see many spot in positive control, PHA (H11 and H12 in the image), but other some wells have many spots. Do you know that PHA is correct as mice splenocytes?

Thank you,

Jun

[Christian@mabtech.com]

Welcome the forum Jun!

Do I understand correctly that you are running this experiment in so called STRIP plates from Millipore? These are difficult to coat by yourself since they are more prone to leakage from the EtOH pre-treatment. In fact, Mabtech does not sell them uncoated, only in precoated format. Your results look like a problem that could occur due to membrane leakage. 

My strong recommendation is that you instead by a Mabtech precoated kit instead. Here is the Mouse IFNg PRO kit:

https://www.mabtech.com/products/elispot-plus-mouse-ifn-g-alp-3321-4ast

I recommend ALP over HRP for ELISpot.

PHA does not work well on mouse cells and we use ConA as a postive control.