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Protocol to activate caspase-1 - Please help!

caspase immunology apoptosis pyroptosis

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7 replies to this topic

#1 JYaron

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Posted 02 July 2013 - 06:31 PM

I've followed a LOT of protocols from the literature and nothing I do seems to be working...

I need to activate caspase-1 to trigger inflammation and pyroptotic cell death.

I'm using THP-1 cells which I differentiate to macrophages using PMA. I use 100 nM PMA and have used a variety of incubation times ranging from overnight to 2 days to 2 days with 4 days of resting in fresh medium, etc.

I think the differentation works because the cells begin to adhere, but remain fairly round. Upon the addition of 1 ug/mL LPS (TCA purified) the cells will start to spread more and a very few of them will display pyroptotic cell death morphology (rounding of the cell membrane, compacting of the nucleus, etc). (see image attached)

I cannot seem to verify the activation of caspase-1 though! I have tried western blotting for IL-1beta maturation and caspase-1 cleavage... I have tried caspsae-1 FLICA (FAM-YVAD-FMK)... I have tried Prizm for caspase-1...

I need a good, tried-and-true protocol for activating caspase-1 so that I can finally move forward with my dissertation project. The literature is a bit too vague, apparently.

Attached Thumbnails

  • lps_treatment.jpg

Jordan Yaron, PhD Candidate
The Biodesign Institute at Arizona State University

www.jordanyaron.com

Ask me about cell biology, microscopy and cell death or find my writing at TheBioBlog.com!

#2 Curtis

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Posted 05 July 2013 - 03:03 AM

I don't see membrane blebbing.

#3 JYaron

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Posted 05 July 2013 - 11:42 PM

I don't see membrane blebbing.


This is true, Curtis. There is no blebbing in this particular picture. Additionally, in caspase-1-dependent cell death the blebbing isn't the same as apoptosis and I only use the word blebbing because there is some level of membrane/cytoskeleton reorganization that occurs. It's more accurately described as sudden membrane rupture followed by partial membrane sealing with cellular swelling by osmotic influx. This results in a round, balloon-like cell with a small, tight circular nucleus. While there isn't on in this picture, there were some that I saw in the population, though the number was probably 1% or less.
Jordan Yaron, PhD Candidate
The Biodesign Institute at Arizona State University

www.jordanyaron.com

Ask me about cell biology, microscopy and cell death or find my writing at TheBioBlog.com!

#4 Curtis

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Posted 06 July 2013 - 04:47 AM

you can check caspase-3 activation or other proteins to make sure cell death pathways are activated. I'm sure you already know about that. According to the picture you posted, I am not sure if what I see is dead cells. Did you run controls?

#5 JYaron

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Posted 09 July 2013 - 12:00 PM

Caspase-3 should NOT be activated, which is the issue. Caspase-1 is the only think I'm looking to be activated and that doesn't necessarily result in cell death. It may result only in the production of mature IL-1beta and IL-18, without cell death.

I have controls, but they look similar to my treatment condition. That's why I'm starting to pull my hair out and I am here to ask for a good, trusted protocol that people have first-hand experience with :/
Jordan Yaron, PhD Candidate
The Biodesign Institute at Arizona State University

www.jordanyaron.com

Ask me about cell biology, microscopy and cell death or find my writing at TheBioBlog.com!

#6 Curtis

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Posted 09 July 2013 - 08:02 PM

Caspase-3 activation is almost the last step of cell death. All different pathways end up with activation of caspase-3. So I'm a little confused why in your experiment it should not be activated.

#7 JYaron

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Posted 10 July 2013 - 09:19 AM

Caspase-1-dependent programmed cell death (known as pyroptosis) is a unique form of cell death that does not depend on the activation of caspase-3. There is detectable activation of caspase-7 downstream, but not 3. Further, DNA degradation in caspase-1-dependent cell death is independent of ICAD cleavage, which is the substrate of caspase-3 and further supports the lack of caspase-3 involvement.

See: http://www.ncbi.nlm....les/PMC2910423/
Jordan Yaron, PhD Candidate
The Biodesign Institute at Arizona State University

www.jordanyaron.com

Ask me about cell biology, microscopy and cell death or find my writing at TheBioBlog.com!

#8 Curtis

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Posted 10 July 2013 - 09:45 AM

Interesting article, so this was discovered just recently. Just one thing, are you sure your antibody is suitable for western blot?





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