Protocols


Screen (pdf version)
ScreenNlrp3 Inflammasome Screen
Posted On02/18/2010 12:24 PM
AuthorOwen M. Siggs
Science WriterNora G. Smart
Background
As in our Toll-like Receptor (TLR) signaling screen, stimulated ex vivo thioglycolate-elicited peritoneal macrophages are used to discover components involved in sensing the adjuvant alum, an aluminium salt that adsorbs and precipitates protein antigens in solution thereby improving vaccine immunogenicity by facilitating the slow release of antigen from the vaccine depot formed at the site of inoculation.  Human PBMC or dendritic cells stimulated with pure TLR4 and TLR2 agonists release only traces of IL-1β, despite inducement of IL-1β mRNA.  In contrast, cells costimulated with TLR agonists plus alum release large amounts of IL-1β due to caspase-1 activation (1).  Alum is now known to activate caspase-1 via the Nlrp3 inflammasome, leading to secretion of the cytokine IL-1β (2).  Nlrp3 is a NOD-like receptor capable of sensing stimuli of microbial origin as well as endogenous markers of cellular damage (3).
 
The alum macrophage screen is designed to probe for additional components of the alum sensing pathway in macrophages isolated from N-ethyl-N-nitrosourea (ENU)-mutagenized mice.  Because inflammasome activation requires two signals for the production of mature IL-1β, macrophages are first primed with lipopolysaccharide (LPS) and then exposed to aluminium adjuvants.  Appropriate macrophage responses to alum are assayed by examining IL-1β levels. 

 

Reagents and Solutions
Brewer’s thioglycolate medium, 4%
4% (w/v) Brewer’s thioglycolate medium powder (BBL Microbiology Systems, Cockeysville, MD) is added to distilled water pre-warmed to 37°C. Solution is autoclaved to sterilize and stored away from light.
 
PEC recovery solution
Hepes-buffered saline solution (Gibco, Invitrogen, Carlsbad, CA )
5% (v/v) heat-inactivated fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA)
200 IU/mL penicillin (Gibco)
200 mg/mL streptomycin (Gibco)
 
PEC medium
Dulbecco’s modified eagle medium (Mediatech Inc., Herndon, VA)
5% (v/v) heat-inactivated fetal bovine serum
200 IU/mL penicillin
200 mg/mL streptomycin
 
MTT solution
2 mg MTT (Sigma)/mL sterile PBS.
 
Lysis solution
90% (v/v) isopropanol
0.5% (w/v) SDS
0.04N HCl
Adjust volume with distilled water.
 
LPS stock
1 mg/ml (Alexis, San Diego, CA, cat # ALX-581-007)
 
Imject Alum stock
40 mg/ml (Pierce, Rockford IL, cat # 77161)
 
IL-1β ELISA (eBioscience, San Diego, CA, cat # 88-7913)
 
PBT
0.5 ml Tween 20 (Sigma)
1L PBS
 
Method
Peritoneal exudate cell (PEC) isolation
  1. Three to four days prior to PEC isolation, 5mL syringes filled with Brewer’s thioglycolate medium are used to inject mice intraperitoneally with 1.5-2mL through a 25-gauge needle.
  2. Immediately prior to isolation, mice are anaesthetized under isofluorane vapour (2-5% v/v, 2% O2).
  3. 5mL syringes filled with sterile PBS are used to recover PECs by lavage through an 18-gauge needle. Once obtained, exudate is added to 5mL of PEC recovery solution in a 15mL conical tube, and stored on ice.
  4. Tubes containing exudate are centrifuged for 5 minutes at 1500 rpm in a tabletop centrifuge, and supernatant is replaced with 1mL of PEC medium. Pelleted cells are resuspended by pipeting, and a 20μL aliquot is taken for cell enumeration.
  5. The concentration of each cell sample is adjusted to 1x106 cells/mL using PEC medium, and 50μL of each sample (5x104 cells) is added to duplicate columns of a tissue culture-treated 96 well flat-bottomed plate, leaving two columns (11 & 12) unoccupied per plate (Table 2). Plates are incubated at 37oC/5%CO2 in a humidified incubator for at least 30 minutes to allow cells to adhere to the plate, during which time LPS solution is prepared.
Activation with LPS/Alum
  1. LPS solution is prepared at 100ng/mL in PEC medium (1μL LPS stock in 10mL PEC medium/plate).
  2. Following preincubation of PECs in the 96 well plate, non-adherent PECs are discarded along with residual medium, and 100μL of LPS solution is added to all wells apart from those in columns 11 & 12. Plates are incubated overnight for at least 16 hours at 37oC/5%CO2.
  3. Following overnight incubation, supernatant is discarded and replaced with 150μL/well of an 800μg/mL Imject Alum solution in PEC medium(300μL Imject Alum stock in 15mL PEC medium/plate), and incubated for a further 8-10 hours at 37oC/5%CO2.
  4. Conditioned supernatant is then collected into another 96 well plate, and retained for IL-1β ELISA.
Table 1 |  IL-1β ELISA plate layout.
 
 
 
 
 
 
 
 
IL-1β
 
 
1
2
3
4
5
6
7
8
9
10
11
12
 
A
sample 1
sample 2
sample 3
sample 4
sample 5
100
 
B
sample 6
etc.
 
 
 
 
 
 
50
 
C
 
 
 
 
 
 
 
 
 
 
25
 
D
 
 
 
 
 
 
 
 
 
 
12.5
 
E
 
 
 
 
 
 
 
 
 
 
6.25
 
F
 
 
 
 
 
 
 
 
 
 
3.125
 
G
 
 
 
 
 
 
 
 
 
 
1.6125
 
H
 
 
 
 
 
 
 
 
 
 
0
 
IL-1β ELISA
  1. On the day prior to assay, 24μL IL-1β capture antibody is diluted in 12mL coating buffer per plate, added at 100μl/well, and incubated overnight at 4°C.
  2. Plates are washed 3 times in PBT.
  3. 200μl/well of assay diluent is added (~20mL/plate), and incubated at room temperature for 1h.
  4. Plates are washed 3 times in PBT.
  5. 1μl of IL-1β standard is added to 1mL of assay diluent (1000pg/mL) for every two plates, and 200μL is added to wells A11 and A12.
  6. A 2-fold serial dilution series is added to rows 11 and 12 (see Table 3).
  7. 125μL PEC-conditioned medium is added to all other wells and incubated at room temperature for 2h.
  8. Plates are washed 5 times in PBT.
  9. 24μL of IL-1β detection antibody is added to 12mL assay diluent per plate, 100μl added to each well and plates are incubated at room temperature for 1 hour.
  10. Plates are washed 5 times in PBT.
  11. 24μL of avidin-HRP is added to 12mL assay diluent per plate, 100μl added to each well and plates are incubated at room temperature for 30 minutes.
  12. Plates are washed 7 times in PBT.
  13. 50μL of TMB substrate solution is added to each well, and incubated away from light at room temperature for 15 minutes.
  14. 25μL of 2N H2SO4 is added to each well, and absorbance is read at 450nm.
Critical Parameters and Troubleshooting
Thioglycolate preparation
Thioglycolate solution should be carefully prepared to ensure maximal PEC recovery.  Solvent should be warmed to 37°C prior to solute addition to assist solubility, and subsequently autoclaved at least once, since this increases inflammatory potential (4).  Aging the solution for at least 1 month also enhances activity (5), in which case it should be stored away from light and at room temperature.
 
Retesting
While stimulated PEC supernatants may be stored indefinitely at -20°C, PECs stored at 4°C remain viable and responsive to stimulation for up to four days.  Prompt completion of the initial assay therefore allows any remaining cells to be used for confirmation of putative mutants.  A minimum of three weeks should be allowed between thioglycolate injections to allow for sufficient PEC recovery.

 

Alleles Identified
Cuties
marula
Nd1
Nd5
Nd6
park1
Park2
Playtar
puff
spark
References
Edit History
Stephen Lyon 11/20/2013 3:21 PM (current)
Nora G. Smart 01/18/2011 2:25 PM