Hi guys,
I tried to transfect poly dA:dT into macrophages to activate Aim2 inflammasome. I setup 4 wells, the first two of which have lps and transfection reagent as negative control. The other 2 wells have lps+transfection reagent and poly dA:dT. I tried both DOTAP and lipoRNAiMAX.
I can see beautiful cleaved form of casp-1 from poly dA:dT transfection. However, I always see cleaved casp-1 in the first two wells. Also, there is a decent amount of IL1b in the supernatant by Elisa.
This is quite confusing, cuz I don't think LPS itself could activate inflammasome. But LPS+transfection reagent always activates inflammasome in my case which is not consistent with publications.
I understand that a 10 hour lps treatment might cause some of the full length il-1b get into the supernatant, so the Elisa will give you some signals. But what's going on with these cleaved casp-1 bands?
I attached a western blot and any help will be appreciated!
thank you!
LPS transfection activates non-canonical NLRP3 inflammasome through Caspase-11. As a control for poly dA:dT transfection, use transfection reagent alone as control.
Dear Deepika Sharma , OMG. I know that LPS will activates non-canonical NLRP3 but I never thought that the transfection reagent would be so potent that it transfects lps used for priming into the cells?
So would you suggest changing fresh medium (no lps) after LPS priming before polydA:dT transfection?
Thank you so much!
I am not sure about the exact procedure being used, but here's what I would do. If the cells need to be primed with LPS (not required for AIM2 activation unless you want to measure IL-1b release), stimulate cells with LPS in RPMI/DMEM media. After priming, wash cells with prewarmed media (RPMI/DMEM) and change media to Opti-MEM (for transfection) and proceed with transfection. This would ensure that there is no contaminating LPS with your transfection reagent. Use appropriate controls- transfection reagent alone (with/without LPS) to make sure you are specifically getting AIM2 activation. Alternately, AIM2 activation can be assessed in unprimed macrophages through caspase-1 cleavage, cell death and IL-18 release. Hope this helps!
Deepika Sharma ,gotcha.
I will give it a try and let you know.
Thank you.
Deepika Sharma , I am completely lost. I washed my cells three times with PBS after LPS priming, however, I can still see casp-1 activation in my control group.
Here's my protocol. Can you help me to point out which might go wrong?
We add L929-conditioned medium (20%) into DMEM/F12 medium to differentiate bone marrow cells into macrophages for 7 days. Then I plate the cells adding 5% L929-conditioned medium into DMEM/F12 as a base. We also add 1%L-glu and 1%HEPES.
Second day after seeding the cells, I prime macrophages with lps and then, after 4 hours, I wash it with PBS for 3 times. I add back DMEM/F12 with L929 and on top of that I add DOTAP only as a negative control.
The only part I think could go wrong is something in DMEM/F12+L929 might be transacted into the cell and activate inflammasome. Maybe I should try opti-MEM as the base for transfection?
Thank you very much.
Xingchen, L929 sup might contain a lot of contaminants. According to the protocol I have used, I cells are stimulated in media without L929 sup for both LPS and transfection. At the time point of stimulation, cells have already undergone differentiation; further since these are relatively short stimulations, growth factors (L929 sup) are not really required during stimulations. Yes, you could also use Opti-MEM as base media for LPS stimuli.
Hope this helps.
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