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

5mg MTT (Sigma)/mL sterile PBS

DMSO

LPS (ALX-581-007-L002)

Flagellin (adipogen, AG-40B-0095-C100)

Lipofectamine 2000 (Invitrogen)

Mouse IL-1b ELISA Ready-SET-Go (eBioscience: 88-7013-76)

Wash Buffer 

1X PBS, 0.05% Tween-20

Stop Solution 

1M H3PO4 or 2N H2SO4

Pipettes

96-well plate (Corning Costar 9018 or NUNC Maxisorp (#44-2404))

96-well ELISA plate reader

DI water

Peritoneal exudate cell (PEC) isolation

1. Three to four days prior to PEC isolation, 3mL 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 3 minutes at 1200 rpm in a tabletop centrifuge, and supernatant is replaced with 1-4mL of PEC medium. Pelleted cells are resuspended by pipeting, and a 10μL aliquot is taken for cell enumeration.

5. The concentration of each cell sample is adjusted to 1x10^6 cells/mL using PEC medium, and 100μL of each sample (1x10^5 cells) is added to a tissue culture-treated 96 well flat-bottomed plate, leaving two columns (11 & 12) unoccupied per plate. Plates are incubated at 37oC/5%CO2 in a humidified incubator for at least 1 h to allow cells to adhere to the plate.

Activation with LPS and flagellin

1. Following preincubation of PECs in the 96 well plate, non-adherent PECs are discarded along with residual medium, and 1 ng/ml of LPS is added to the cells. Plates are incubated for a further 4 hours, during which time flagellin solution is prepared.

2. 70 ng flagellin is mixed with 25μL OptiMEM medium per well and mixed gently.

3. 1 μL Lipofectamine 2000 is mixed with 25μL OptiMEM medium per sample to be assayed, mixed gently and incubated at room temperature for five minutes.

4. Lipofectamine and flagellin solutions are gently mixed in one tube, and incubated at room temperature for twenty minutes.

5. 50μL of Lipofectamine/flagellin mix is added to all sample wells of the 96 well plate, and plates are incubated for 2 hours.

6. Conditioned supernatant is collected into another 96 well plate and retained for IL-1b ELISA Assay. The cells are replaced with 100μL/well of a 1:4 MTT solution:PEC medium solution (MTT 1mg/mL final concentration). PEC plates are incubated at 37oC/5%CO2 overnight. Supernatants are removed and 100 ul DMSO is added to each well, shake the plate for 10 min and read plate at 570 nM. This value is used as cell number normalization for the ELISA results.

IL-1β ELISA

1. Coat ELISA plate with 100 μL/well of capture antibody in Coating Buffer. Seal the plate and incubate overnight at 4°C.

2. Aspirate wells and wash 3X with >250 μL/well Wash Buffer. Allow time for soaking (~1 minute). Blot plate on absorbant paper to remove residual buffer.

3. Dilute 1 part 5X concentrated Assay Diluent with 4 parts DI water. Block wells with 200 μL/well of 1X Assay Diluent. Incubate at room temperature for 1 hour.

4. Optional: Aspirate and wash at least once with Wash Buffer.

5. Using 1X Assay Diluent, dilute standards as noted on the C of A to prepare the top concentration of the standard. Add 100 μL/well of top standard concentration to the appropriate wells. Perform 2-fold serial dilutions of the top standards to make the standard curve for a total of 8 points. Add 65 μL/well assay diluent plus 35 ul samples to the wells. Seal the plate and incubate at room temperature for 2 hours (or overnight at 4°C)

6. Aspirate/wash as in step 6 of the IL-1b ELISA protocol. Repeat for a total of 3-5 washes.

7. Add 100 μL/well of detection antibody diluted in 1X Assay diluent. Seal the plate and incubate at room temperature for 1 hour.

8. Aspirate/wash as in step 6 of the IL-1b ELISA protocol. Repeat for a total of 3-5 washes.

9. Add 100 μL/well of Avidin-HRP diluted in 1X Assay diluent. Seal the plate and incubate at room temperature for 30 minutes.

10. Aspirate and wash as in step 6 of the IL-1b ELISA protocol. In this wash step, soak the Wash Buffer for 1 to 2 mintues prior to aspiration. Repeat for a total of 5-7 washes.

11. Add 100 μL/well of Substrate Solution to each well. Incubate plate at room temperature for 2-3 minutes.

12. Add 50 μL/well of Stop Solution,

13. Read plate at 450 nm.