Ab142085

For activation by the TLR2 agonist (17 (link)), subconfluent BEEC monolayers were pre-exposed to pam3Cys-Ser-(Lys)4, a synthetic analog of the triacylated N-terminal part of bacterial PGN (ab142085, Abcam) (10 pg ml⁻¹), for 24 h followed by co-culture with sperm for 6 h. For blocking by the TLR2 antagonist (20 (link)), BEECs were pre-incubated with CU-CPT22, a synthetic TLR2 blocker (Merck, Darmstadt, Germany) (0.1 μM = 36.24 ng ml⁻¹), for 3 h before being exposed to PGN (10 pg ml⁻¹) for 24 h followed by co-culture with sperm for 6 h.

In order to elucidate the heterodimeric form of TLR2 signaling in sperm- induced inflammation in bovine uterus at cellular level, BEECs were co-cultured with sperm (5 million/mL) for 3h. Furthermore, to investigate the contribution of TLR2 signaling (TLR2/1 and TLR2/6) cascade to uterine inflammation, the following experiments was conducted using different TLR2 agonists. Bovine endometrial epithelial cells (BEECs) were stimulated with PAM3 (TLR2/1 agonist, ab142085, Abcam) and PAM2 (TLR2/6 agonist, InvivoGen, USA) at 100 ng/mL concentration for 1h. The concentration of dose and time point were selected based on our previous reports in which those conditions were investigated in detail (2 (link), 7 (link)). In brief, based on dose- and time-dependent investigations (10⁴, 10⁵ and 10⁶ sperm/mL), 5 million/mL of sperm was used to induce the weak physiological inflammation after co-culture with BEECs for 3 h (8 (link)). As well, PAM3 (10, 100 and 1000 ng/mL) was applied, and 100 ng/ml was the first to induce the inflammatory response in BEECs at 1h of incubation in the similar level to that of sperm (2 (link)). Thus, 100 ng/mL of PAM3 and the same concentration of PAM2, were used in the present study to compare their inflammatory effects with sperm.