Hi guys, I am trying to see inflammasome activation after delivering flagellin from salmonella into macrophages. I use DOTAP transfection reagent from Roche which is widely used by other publications. However, I could not see any cleaved caspase-1 band.
The antibody is definitely good, cuz I can easily visualize the cleaved caspase-1 in salmonella infection. Moreover, I can tell the full-length caspase-1 decreases in flagellin-transfected macrophages, however, there is not cleaved form.
This really gives me a headache and any help will be appreciated!
Thank you all in advance!
Alex Kashkin
,thank you Alex and I will look into this paper right away!
Can you provide more details on your protocol? What cell type? Are you concentrating your supernatant? What antibody? Lots of variables.
Jordan R. Yaron Dear Jordan, Sure, here are some details:
I isolated and differentiated bone marrow into macrophages using L292 as supplement and dmem-f12 as basic medium. After about a week, I plated them into 6-well plates and transfected flagellin into BMDM( primed with LPS for 4 hours) using DOTAP. Six hours later, I collected the supernatant for elisa and also the cells for western blot.
What I looked at are both full length and active caspase-1 in the cells, also the mature IL-1B in the supernatant.
I usually see really strong caspase-1 activation in the cells with Salmonella infection. However, I could not see this when I transfected flagellin from salmonella into the cells.
I am troubleshooting right now, one possible reason would be the the purity of the flagellin we purchase is only 10% which is kinda low. So when I transfected 2 ug of flagellin, there might be only like 0.2ug in it.
Caspase-1 antibody I used is from adipogene which is probably the best out there.
Hopefully, this will give you a better idea.
Thank you again,
Xingchen
Hi. This kind of experiment are quite tricky and are very cell type dependant... We usually only get signal by using very high amout of Flagellin. The easiest really is to get some Fla-Tox from Russel labs.... (We are in the process of making it and we should have some commercial option within a couple of months).
Eric Perouzel That's true. my advisor purchased flagellin from invivogen which is the one with only about 10% purity. I transfected about 10ug into the macrophages and finally i started to see a weak band of cleaved caspase-1. But still the poly da:dt transfection is driving me nuts. Thank you for ur answer.
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