Hi! I am doing ELISPOT for the first time has no previous experience. I want to test mouse spleen cells injected with peptides. Right now I am trying to make my positive control work. I am using PVDF membrane from Millipore, type MSIP which requires 15 ul of 35 % ethanol treatment for 1 min.
I am using purified anti-mouse IFN- gamma from Biolegend # XMG1.2 for coating at 15 ug/ml ON at 4 C.
Next day taking out spleen cells from Liq N2, counting them and seeding around 10^5 or 10^4 cells / well.
Stimulating cells with concanavalin for 24 hrs -more than 48 hrs at 5 ug/ml-500 ug/ml
Wash and then add Biotin -anti mouse IFN gamma (Biolegend # 505704) at 1 - 2- 10 ug/ml for 2 hrs at 37 C
Wash and add HRP-Streptavidin from BD # 557630 at 1:100 for 1 hr at 37 C
Wash and add AEC substrate set from BD # 551951
wait till 30 min or more and then wash with water.
But do not see enough spots like ony 10 or so and as I am doing in duplicates it is not replicated the other well would not show any spot . Sometimes more cells 10^6 show results and sometimes less cells 10^4
Basically there is no consistency, few spots and also there is high background.
Pl. suggest what to do. Where I am going wrong?
Thanks!
Puja
Do you ever tried fresh spleen cells ? If you see counts your reagents are working. ConA is in my hands fine to stimulate IFNg (often more than 700 spots when using 2 x105 cells. I think you frozen spleen cells might be the problem (I have never done this or heard about such a protocol).
Best Thomas
Hi Puja
The best positive control is immunize the mice with OVA (plus adjuvant like poly(I:C) and stimulate the spleen cells with SIINFEKL, if you want a specific immune response positive control. And remember ... if you don´t administrate the peptides with any adjuvant ... is almost impossible detect IFN-g by ELISPOT.
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