Hi, everyone.
I`m trying to activate NLRP3 inflammasome in various cell types with classical method (LPS + ATP).
While I reviewed this process, I noticed that the concentration of ATP I`m using (2mM or 5mM, which is considered as normal to induce NLRP3 inflammasome activation in many papers) is much higher than the concentration that is known to be cytotoxic (less than 100uM...).
I cannot find any difference in viability between control and LPS+ATP treated cells.
How is it possible? Is it LPS priming that protect cells from ATP-induced cytotoxicity? Or Do I misunderstand the inflammasome activation process/ ATP-induced cell damage? Please help me.
Could you provide more information about your experimental system?
What cell type are you using? What concentration and duration of time are you using for LPS and ATP? What is your measurement of cell death? (LDH? PI?)
Hi, thanks for your reply.
I`m using primarily cultured microglia derived from neonate mice.
To induce NLRP3 activation, LPS(100ng/ml) is pretreated for 6 to 24 hrs then ATP (2mM) is added to the medium. Actually I have not tested whether this procedure activates NLRP3 inflammasome with IL1beta secretion yet (I`m planning to) but it is widely used protocol so I followed.
My major focus is on the inflammasome thing so I did not check cell damage with any specific methods after these treatment but morphology of microglia was not changed and no sign of cell detachment was not observed through the microscope.
How long is the ATP added for and how are you preparing your ATP solutions? What organism is your LPS from? You can do a western blot for intracellular pro-IL1b induction to determine if your priming step is the issue. I do 1 ug/mL for 4 hours and get robust induction as determined by both WB and IF.
I would recommend at least 30-60 minutes of ATP exposure. You can use a membrane-impermeant dye like ethidium bromide to determine if the P2X7 receptors are being activated by the ATP. I would recommend doing immunofluorescence for ASC or Caspase-1 or YVAD-FLICA to see if you have perinuclear specks that are indicative of inflammasomes.
5 mM ATP for 30-60 minutes will kill your cells, for sure. You can use a caspase-1 inhibitor like YVAD-CHO or YVAD-CMK and if you see a reduction in death then you are good to go.
Westerns on concentrated supernatants for IL1b or Caspase-1 are also a sure-sign, or ELISA for IL1b.
Hi Yoojin,
Regardin the difference between ATP cytotoxicity and NLRP3 inflammasome activation, the processes are actually closely linked. When you treat LPS-primed cells with 5 mM ATP to trigger NLRP3 activation, the cytokine release (IL-1b and IL-18) is accompanied by ATP-induced permeabilization of the cell membrane and after prolonged stimulation, a form of caspase-1-dependent proinflammatory cell death named pyroptosis.
I use 4h LPS priming (1 ug/ml) followed by 30-45 min treatment with 5 mM ATP to activate the NLRP3 inflammasome in primary human monocyte-derived macrophages. I start to see clear rounding of the cells and loosening of their adherence to substratum already around 15-20 min of ATP treatment and after 45 min, the rounding is evident in virtually all the cells.
I was wondering how you prepare your ATP stock solutions? I prepare a fresh, neutralized 400-500 mM stock solution of ATP in cell culture medium immediately before use in experiments. From what I understand, ATP stock solutions can also be stored at -80 C for some time, but they are quite unstable and for example prolonged storage at +4 C might cause problems.
Hope you get your protocol running soon!
- Badges
- Science topic
- Similar topics
- Methodology
- Laboratory
- Cellular Biology Techniques