|Screen||TLR Signaling Screen|
|Posted On||08/12/2010 7:29 AM|
|Author||Owen M. Siggs|
The Toll-like receptors (TLRs), attentive to the molecular themes of microbial infection, constitute a key sensory apparatus of the innate immune system (1). Ten TLRs are present in man, twelve in mouse, with a total of thirteen between both species. The signaling capacity of seven of these are monitored in our screen (Table 1) through the ex vivo stimulation of thioglycolate-elicited peritoneal cells.
Table 1 | TLR Ligands.
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).
|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)
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
Dulbecco’s modified eagle medium
10% (v/v) heat-inactivated fetal bovine serum
200 IU/mL penicillin
200 mg/mL streptomycin
10mg/mL cycloheximide (Sigma) in sterile PBS, and diluted 1:25 in L-929 medium immediately prior to use.
2mg MTT (Sigma)/mL sterile PBS.
90% (v/v) isopropanol
0.5% (w/v) SDS
Adjust volume with distilled water.
Peritoneal exudate cell (PEC) isolation
Activation with TLR ligands
Table 2 | 96 well plate layout.
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.
|Critical Parameters and Troubleshooting|
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 (11). Aging the solution for at least 1 month also enhances activity (12), in which case it should be stored away from light and at room temperature.
Upon receipt, new ligand stock should immediately be distributed into individual aliquots and stored at -70°C. Each ligand should then be tested for purity using PECs from mice deficient in the corresponding TLR, to ensure that no TNF is produced in its absence.
L-929 cell renewal
Once received from source, L-929 cells should first be tested for sensitivity to TNF. Once confirmed, sensitive cells should be expanded and stored in liquid nitrogen in multiple aliquots. Since L-929 cells lose their sensitivity to TNF over time, these aliquots may be thawed as required.
While stimulated PEC supernatants may be stored indefinitely at -20°C, PECs stored at 4°C remain viable and responsive to TLR stimulation for up to four days. Prompt completion of the initial L-929 bioassay 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.
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