ASC crosslinking always working for us. We lysed cells, often including shearing through G22 needle, spin lysate down, remove sup, resuspend pellet in 200 ul either PBS or RPMI (pellet is still visible), add freshly prepared DSS (1-2 mM final concentration) for 30 min at room temperature, then quench with Tris-HCl (20-50 mM final), spin pellet down and directly resuspend in Laemmli buffer. We use our own home made polyclonal Ab and secondary DAR Ab.

Dear Mikhail A Gavrilin ,

I actually also tried a protocol close to yours.

Here it is.

1. Detach cells by scraping in 1 ml ice cold PBS containing 2 mM EDTA and centrifuge for 5 min at 1,500 x g at 4 °C.

2. Resuspend cell pellets in 0.5 ml of ice-cold buffer A and lyse by shearing 30 times through a 21-gauge needle in microcentrifuge tubes. Centrifuge lysates for 8 min at 1,800 x g 4 °C to remove bulk nuclei. Keep 30 μl of lysates for Western blots of ASC as input controls.

3. Dilute the remaining supernatants in a 1:1 ratio with buffer A and centrifuge at 2,000 x g for 5 min at 4 °C. After centrifugation, collect and dilute supernatants with 1 volume of CHAPS buffer and centrifuge at 5,000 x g for 8 min to pellet ASC oligomers.

Here comes the problem for me. They mentioned they resuspend pellet with CHAPS buffer in step 3. However, I did not see any visible pellet.

4. Discard supernatants and resuspend pellets in 50 μl of CHAPS buffer containing 4 mM of disuccinimidyl suberate (dissolved in DMSO). Incubate for 30 min at room temperature to cross-link proteins.

Buffer A 20 mM HEPES-KOH, pH 7.5 10 mM KCl 1.5 mM MgCl2 1 mM EDTA 1 mM EGTA 320 mM sucrose CHAPS buffer 20 mM HEPES-KOH, pH 7.5 5 mM MgCl2 0.5 mM EGTA 0.1 mM PMSF 0.1% CHAPS

Quick question, when you say the pellet is still visible, do you mean you can crosslink the pellet without break it?

Thank you very much!

Xingchen

Mikhail A Gavrilin - Out of curiosity, what is your lysis buffer recipe in your ASC crosslinking experiments?

When we do in vitro ASC specks assembly (Alnemri method), we use CHAPS buffer supplemented with protease inhibitors. ASC should be concentrated (30x10^6 cells lysed in 100 ul CHAPS, span for 10 min at 16,000 g, then 40 ul aliquot is incubated at =37C and 40 ul on ice. Then we add 360 ul of PBS (1:10 dilution of the original  lysate), divide: 200 ul without DSS and 200 ul with DSS for 30 min at room t. Then spin down, collect sup and resuspend pellet in similar volume of PBS. Then 20 ul loaded on gel (see new picture)

When we use stimulated cells (in cells ASC oligomerization), we lyse cell pellet in buffer A (20mM HEPES-KOH, pH 7.4, 150nM KCl, 1% IGEPAL CA-630), spin down and separate cell extract from insoluble pellet, wash insoluble pellet with PBS, then resuspend in PBS, add DSS room t for 30 min, spin down, separate supernatant from pellet, resuspend pellet in 20 ul Laemmli, run gel - see ASC oligomers.

Remember: Tris containing buffer is not good because Tris quenches DSS crosslinking reaction!

Xingchen,

When we do in vitro speck assembly, we spin down all insoluble pellet first and then monitor ASC formation in sup at 37C. Side control is at 4C - monomers, no specks.

When we lyse cells to see ASC assembly in cell as a result of stimulation, we do not prespin down bulk nuclei because ASC specks are trapped in cell matrix and also pelleted. We have nice side control to monitor every step - our YFP-ASC THP-1 cells and fluorescent microscope. We spin down cell lysate only once after lysis on ice, and separate insoluble pellet from sups. So, you may already pellet your ASC specks before crosslinking.

And yes, we add DSS to insoluble pellet, vortex it periodically during 30 min incubation at room temperature, then spin down and resuspend insoluble pellet in Laemmli.

Dear Mikhail A Gavrilin ,

Thank you so much!

I work on BMDMs and stimulated with lps+atp for inflammasome activation.

Maybe TBS buffer interferes with DSS crosslinking and that's why some people wash pellet with PBS after lysing cells with TBS.

According to your detailed protocol, now I am sure that my own protocol should work since it looks similar to yours. The only thing I would change is switching to PSB to wash and resuspend the pellet before DSS crosslinking.

My major confusion previously was if the crosslink would work on an insoluble pellet. Now I feel confident to proceed and I will let you know when I get my results.

Thank you very much!

Best,

Xingchen

Thank you Mikhail A Gavrilin for the thorough and explicitly clear protocol :)

Good luck, Xingchen. It works as a charm.

Hi Mikhail and xinchen,@Mikhail @ Xingchen Dong

when I add DSS(disolved in DMSO first) into the PBS, it separated out from PBS immediatly. After crosslinking , DSS was spin down with ASC. The problem is DSS is insoluble, it has to be loaded in the SDS-PAGE with crosslinked protein. Then, the insloluble DSS jam the gel, disturbe the seperation of proteins.How can I solve this problem?

We add 1 ul of DSS in DMSO to 25 ul of ASC solution in PBS And see no problems with ASC oligomerization and SDS-PAGE with WB.

Thanks Mikhail, finnally ,I found simply heat PBS contained DSS at 100℃ for 10min, the white precipitate would dissappear!

But I still have a question , which kind of SDS protein loading buffer should I ues, the reduced buffer(contained DTT) or non-reduced buffer? I notice this paper mentioned that non-reduced buffer should be used. Many thanks to Mikhail A Gavrilin Mikhail

https://www.pnas.org/content/113/32/E4671.long#sec-10

AIM2 inflammasome is activated by pharmacological disruption of nuclear envelope integrity.

Proc Natl Acad Sci U S A. 2016 Aug 9;113(32):E4671-80. doi: 10.1073/pnas.1602419113. Epub 2016 Jul 26.

We use Laemmli buffer with 2-ME (4x) and of course boil samples for 5-10 min at 95 C

We do cross linking at room temperature. DMSO may precipitate at lower temperature.

really appreciate to Mikhail for your suggestions, I will try again according to your kindly guide.Many thanks!@Mikhail