BIE cells were plated at 3×104 cells/well of a 12-well ptype I collagen-coated plates (Iwaki, Tokyo, Japan), and cultured for three days. After changing medium, lactobacilli (5×107

cells/ml) were added and 48 hours later, each well was washed vigorously with medium at least 3 times to eliminate all the stimulants. Expression of cytokines, chemokines and TLRs negative regulators were studied first without any inflammatory challenge by using real time PCR as described below. find more In addition, the effect of lactobacilli on BIE cells immune response was studied using heat-stable ETEC as inflammatory factor. BIE cells were treated with heat-stable ETEC (final concentration: 5×107 cells/ml) for indicated time and the expression of cytokines, chemokines and TLRs negative regulators were studied by using real time PCR as described below. In addition, activation of p38, c-Jun N-terminal kinase (JNK) and

extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases and NF-кB pathways were studied by using western blotting as described Clomifene below. In these experiments, the synthetic TLR2 agonist selleck kinase inhibitor tripalmitoylated lipopeptide Pam3CysSerLys4 (Pam3CSK4) was also used. BIE cells were stimulated with Pam3CSK4 (final concentration: 200 ng/ml) for the indicated time same as the other stimuli. Quantitative expression analysis of cytokines, chemokines and TLRs negative

regulators by PCR in BIE cells Two-step real-time quantitative PCR was used to characterize the expression of cytokines, chemokines and TLRs negative regulators mRNAs in BIE cells. Total RNA from each sample was isolated from the BIE cells using TRIzol reagent (Invitrogen). All cDNAs were synthesized from 5 μg of total RNA using a Quantitect Reverse Transcription kit (Qiagen, Tokyo, Japan) according to the manufacturer’s recommendations. Real-time quantitative PCR was carried out using a 7300 Real-time PCR System (Applied Biosystems, Warrington, UK) using Platinum SYBR Green qPCR SuperMix UDG with ROX (Invitrogen). The primers for cytokines, chemokines and TLRs negative regulators used in this study are described in Table 1.