Protocol for MSU crystals activation of the NLRP3 inflammasome

Pablo P.Group Leader at Hospital Virgen de la Arrixaca

Activation of the NLRP3 inflammasome by MSU crystals is a well
established method to induce the release of mature IL-1beta from
LPS-primed macrophages. However, we have experienced many
difficulties to achieve this, and found differential response by using
different macrophage types (THP1, mouse BMDMs, mouse peritoneal
macrophages...), type of MSU crystals used (home made vs
purchased), stimulation time... and usually this protocol ends up with
very high levels of LDH release.
I would like to share different protocols used across different labs
(type of macrophages, stimulation conditions, MSU brand...) in order
to discuss which are the ideal conditions to get this protocol running.

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February 12, 2013
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Sbastien R., Gonzalo D. like this
3 comments

Pablo
Pablo P.
Group Leader at Hospital Virgen de la Arrixaca
Our current protocol:
-

Mouse BMDMs at a density of 0.42 x 10^6 cells/cm2

Allow 4-6 h adherence to the substrate and stimulate with LPS (1 ug/ml) for 4h
Wash LPS 3x with optimem
Add MSU crystals (200 ug/ml - ALX-400-047-M002 - Enzo Life Sciences) in optimem
Incubate 16 h
Collect supernatants and analyze IL-1beta by ELISA and WBs

Hope this will help!

George
George D.
Professor at Case Western Reserve University

We've been studying NLRP3 inflammasome activation by phagocytosed alum in both BMDM
and BMDC. Like Sebastien, we also prime with LPS (1 ug/ml) in low serum (0.1%) DMEM for 4
hrs. We stimulate with imject alum (400-500 ug/ml and brief centrifugation to deposit the
alum crystals on the adherent macs or DCs). Release of processed IL-1b starts after an ~1.5
hr lag and then progressively increases over the next 2-4 hrs. LDH release starts at ~3-4 hr.
So, we typically stimulate with alum for 3 hrs to obtain robust IL-1b release but minimal LDH
release.

Gloria
Gloria L.
MCCIR- Prize Postdoctoral Fellow
The protocol we use:
- Mouse peritoneal macrophages at a density of 0.5 x 10^6 cells (in 24 well plate) allow to
adhere o/n. THP-1 cells (0.5 x 10^6 cells (in 24 well plate)) are stimulated with PMA 0.5uM
for 3 h and then incubated with fresh media o/n.
- Stimulate cells with LPS (1 ug/ml) for 2h in RPMI 10% serum for peritoneal macrophages o
4h in DMEM, 10% serum for THP-1.
- Wash LPS 1x with PBS
- Add MSU or CPPD crystals (250 ug/ml - Invivogen) in RPMI or DMEM with no serum.
- Incubate 1h.
- Collect supernatants and analyze IL-1beta by ELISA and WBs.