In the literature it is either unclear or not mentioned what people do and if there is a consensus of how to do this.
From what I have seen, when you prime with LPS, you are in complete media but then you change media for without FBS before adding ATP. Is that correct?
My second question is: once you're done priming with LPS do you change media before pulsing with ATP (if you are using complete media all the way through)?
Thanks!
Normally, preparation of immortalized microglial cells from caspase-1-deficient and IL-1R-deficient mice and macrophages from NLRP3-deficient mice and ASC-deficient mice and corresponding wild-type control (C57BL/6) have been described by Halle et al. and Hornung et al. Immortalized microglial cells, immortalized macrophages and RAW-Blue cells are cultured in DMEM supplemented with 10% FBS. CAD cell line is cultured in Optimem medium supplemented with 10% FBS. No it is not correct when prime with LPS, material is transferred in complete media, You chould 10% FBS before adding ATP.
We have activated NLRP3 inflammasome with with its inducers such as nigericin or ATP and Aim2 inflammasomes with synthetic DNA in LPS-primed THP-1 cells in a complete medium. See our papers.
Excellent question! It depends on what your method to assess the inflammasome is. If you are measuring IL1b or IL18 readout by ELISA or cell death by LDH then you can use complete medium for all steps. If, on the other hand, you are doing a Western Blot then it is generally recommended to use serum-free medium because detection of activated caspase-1 and processed IL1b and IL18 in the supernatant have to be concentrated (by your favorite method) and serum will overload your concentrated sample. I usually wash once with serum-free medium and then add serum-free medium with my stimulus.
Be aware, however, that some stimuli take many hours to take effect and serum starvation can cause off target responses. In these cases it is sometimes effective to use a very low concentration of serum (1% or less) simply to keep your cells alive. You might still run the risk of overloading the concentration process, so you will have to optimize it according to your protocol.
For ATP, since that's the example you used, you should be OK with serum-free conditions because the effect is quite fast (30-60 minutes for very robust response).
I agree with Jordan's recommendations with respect to the ability to detect secreted proteins in the medium. In our cell system, we needed to optimize the protein concentration step to detect secreted proteins in the medium that had 10% serum.
Thanks Jordan. That is exactly what I want to do, to look in the medium by WB.... I tried that yesterday, let's see after precipitation if I can get something because the protein concentration was very low...
Thanks everyone or your comment. I will have a look at your papers.
Inflammasome release ( including IL-1beta and IL-18) is greatly reduced in the absence of FBS. LPS and ATP are working fine in the presence of FBS. If you are measuring IL-18 release, LPS priming is sufficient for 5 minutes before ATP pulsing ( See Our last article in JI by Ghonime M et. Al)
@Mikhail are you measuring it by WB or ELISA? In THP-1 cells too?
Thanks!
Both, WB and ELISA. In THP-1 cells too. Usually we clearly see released cleaved p20 caspase-1 when IL-1 beta is around 5 ng/ ml and more ( 300 pg/ ml for IL-18). We have our home made Ab for IL-1 beta and caspase-1. ASC is very abundant and well detected by WB and ELISA. Also, I would suggest to check THP-1 on mycoplasma contamination because it may affect inflammasome release ( see our article in PLOS ONE, 2010).
We have also done the ATP stimulation in complete media and measured IL-1 beta by ELISA. It does make sense to use SFM or low serum conditions for blots.
Hi Emilie Degagné,
Have you solve this problem? How did you concentrated proteins in medium? Thanks?
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