Fla st ultrapure

FLA-ST Ultrapure (purified flagellin from Salmonella Typhimurium, a TLR5 agonist) and the vehicle solution, endotoxin-free water, were purchased from InvivoGen (San Diego, CA, USA). In this experiment, it was applied at 0.1 μg, 0.3 μg and 0.9 μg in 10 μL vehicle solution separately, which was modified from the previous procedure of intraplantar administration with flagellin [19 (link)].
Go 6976 was purchased from Tocris Bioscience (Ellisville, MO, USA) and prepared in 0.1% DMSO in 0.1 M PB [57 (link)]. FLA-ST Ultrapure 0.9 μg (InvivoGen, San Diego, CA, USA) was used to combine with Go 6976, which separated into four subgroups (0 μM, 5 μM, 20 μM and 50 μM; n = 3 per subgroup) respectively. Likewise, DAMGO 25 μg (Tocris Bioscience, Ellisville, MO, USA) was used to combine with Go 6976 and also assigned into four subgroups separately.

Purified recombinant flagellin from Salmonella Typhimurium (FLA-ST Ultrapure) and loxoribine were purchased from InvivoGen (San Diego, CA, USA). Lipopolysaccharide (LPS) was purchased from Enzo Life Sciences (Lörrach, Germany). LY294002 was obtained from Cell Signaling Technology (Danvers, MA, USA), while wortmannin and rapamycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Akt inhibitor IV was obtained from Calbiochem (San Diego, CA, USA). LY294002, wortmannin, and rapamycin were solved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA). In all experiments using the inhibitors, DMSO-containing DMEM medium complete (see below; DMSO dilution at 1:1000 vol/vol) served as negative control. Anti-mTLR5 neutralizing IgG antibody was obtained from InvivoGen.

To study the activity of TLR3 and TLR5 in D-5 human embryos, their specific ligands poly (I: C) (InvivoGen, San Diego, CA, USA) and flagellin (FLA-ST Ultrapure, from Salmonellatyphimurium, InvivoGen, San Diego, CA, USA), were added to embryo culture media. Fifteen human embryos at pronucleate (PN) or early cleavage (2–4-cell; EC) stage were thawed on two different occasions and cultured in G1 media (Vitrolife, Gothenburg, Sweden) to D-3 and G2 media (Vitrolife, Gothenburg, Sweden) to D-5. D-5 blastocysts were then treated with 0.5 or 1 μg/ml poly (I: C), 50 or 100 ng/ml flagellin, or G2 medium alone (control) for 24 h. Poly (I: C) and flagellin concentrations used were taken from the literature (Le Tortorec et al., 2008 (link); Aboussahoud et al., 2010 (link)), as well as the manufacturers’ instructions, taking into consideration that poly (I: C) is a potent TLR3 activator. D-6 blastocysts were lysed for PolyAPCR (see below), while the 24 h supernatants were collected, centrifuged at 10,000g for 5 min at 4°C, transferred to fresh tubes and stored at −80°C.