TLRs are potent inducers of the proinflammatory cytokine tumor necrosis factor alpha (TNFα), and to this end TNF is used as our screening endpoint. Bioactive TNF (α and β) is measured by virtue of its cytotoxicity towards the mouse fibroblast line, L-929 (9) (American Type Culture Collection: CCL-1), and since TNFβ expression is restricted to lymphoid cells, all TNF bioactivity derived from purified macrophages will be due to TNFα. L-929 viability is determined by the addition of the yellow tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), which is reduced to a purple formazan product through cleavage of the tetrazolium ring. Since this reaction occurs only in the mitochondria of live cells, viability may be inferred colorimetrically (10).

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 37^oC/5%CO₂ in a humidified incubator for at least 30 minutes to allow cells to adhere to the plate, during which time TLR ligand solutions are prepared.

6. TLR ligand solutions are prepared at predetermined concentrations in PEC medium, using the minimum concentration necessary to elicit a maximal TNFα response by L-929 bioassay (Table 1).
7. Following preincubation of PECs in the 96 well plate, non-adherent PECs are discarded along with residual medium, and 100μL of each TLR ligand solution is added to each well of the corresponding row (Table 2). Plates are incubated for a further 4 hours.
8. Conditioned supernatant is collected into another 96 well plate, and replaced with 100μL/well of a 1:4 MTT solution:PEC medium solution (MTT 400mg/mL final concentration). PEC plates are incubated at 37^oC/5%CO₂ overnight, and conditioned supernatant retained for TNF bioassay.

9. On the day prior to bioassay, 5x10⁴ L-929 cells are added to every well of a tissue culture-treated 96 well flat-bottomed plate in a volume of 100μL L-929 medium, and incubated overnight at 37^oC/5%CO₂.
10. Immediately prior to bioassay, supernatant is replaced with 50μL/well of cycloheximide solution, and incubated at 37^oC/5%CO₂ for at least 20 minutes.
11. A mouse recombinant TNFα (ALX-522-009, Alexis, San Diego, CA) standard series is prepared in the interim, using 7 serial two-fold dilutions (starting from ~50pg/mL) and L-929 medium alone. 50μL of each standard point is added in duplicate to columns 11 & 12 of each L-929 plate (organized as in Table 2), and 50μL of L-929 medium alone to rows 1-10. Finally, 5mL of conditioned PEC medium is added to the corresponding wells of each L-929 plate, and plates are incubated at 37^oC/5%CO₂ for 12-16 hours.
12. Following incubation, 25μL/well of MTT solution (400mg/mL final concentration) is added to the L-929 plates, which are then incubated at 37^oC/5%CO₂ for an additional hour.
13. Supernatant from all plates (L-929 and PEC) is discarded and replaced with 80μL/well lysis solution, then incubated at room temperature for 30 minutes to allow the purple formazan precipitate to enter solution.

Absorbance at 590 nm is read. For L-929 plates, bioactive TNF concentrations are inferred from the standard curve of [TNFα] versus absorbance. For PEC plates, the absorbance values provide an indication of the number of live cells used in the TNF assay. To adjust for sample to sample variation in PEC number (which may affect the amount of TNFα produced), the calculated TNF concentrations must be normalized. Average absorbance of each PEC sample (e.g. average absorbance of columns 1 and 2, 3 and 4, 5 and 6, etc. on PEC plate) is divided by the total average absorbance across all PEC plates. The resulting figure is used to normalize each TNF concentration value. Typically, samples falling outside three standard deviations of the mean are considered putative mutants, and leftover PEC solution may be used for ligand dose-response retesting.