6 ± 1.1) [60]. The exponential and stationary phase cells were washed twice in phosphate buffered saline (PBS) at pH 7.4, suspended at 2 × 108 cells/ml in PBS containing 20% glycerol, and stored at -80°C until co-culturing with Thiazovivin human immune cells. Quantification of the exponential and stationary phase viable cells before and after freezing showed no significant losses in cell viability (data not shown). Colony forming units (CFUs) were determined by plating serial dilutions of the cultures on MRS agar (data not shown). BAY 80-6946 molecular weight Peripheral blood mononuclear cells assay This study was approved by Wageningen University Ethical

Committee and was performed according to the principles of the Declaration of Helsinki. Peripheral blood of healthy donors was from the Sanquin Blood

Bank, Nijmegen, The Netherlands. Before sample collection, a written informed consent was provided. Peripheral blood mononuclear cells (PBMCs) were separated from the blood of healthy donors using Ficoll-Paque Plus gradient centrifugation according to the manufacturer’s protocol (Amersham biosciences, Uppsala, Sweden). After centrifugation the mononuclear cells were collected, washed in Iscove’s Modified Dulbecco’s Medium (IMDM) + glutamax (Invitrogen, Breda, The Netherlands) and adjusted to 1 × 106 cells/ml in IMDM + glutamax supplemented Anlotinib purchase with penicillin (100 U/ml) (Invitrogen), streptomycin (100 μg/ml) (Invitrogen), and 1% human AB serum (Lonza, Basel, Switzerland). PBMCs (1 × 106 cells/well) were seeded in 48-well tissue culture plates. After an overnight rest at 37°C in 5% CO2, 5 μl aliquots of thawed bacterial suspensions at 2 × 108 CFU/ml were added to the PBMCs (L. plantarum: PBMC ratio of 1:1). PBS (5 μl) and LPS (1 μg) served as negative (PBS) and positive (LPS, TLR4 ligand) controls for the stimulation of PBMCs. IL-10 was produced in sufficient amounts for quantification in response to LPS but not to PBS. Similarly, neither LPS nor the PBS buffer stimulated the GNAT2 production of IL-12. To test the capacity of the 42 L. plantarum strains to stimulate PBMC cytokine

production, PBMCs from 3 different donors were examined (donors A, B, and C). For donors A and B, separate stationary-phase cultures of each L. plantarum strain were used. For donor C, both replicate cultures of each L. plantarum strain were examined. In PBMC assays comparing responses to L. plantarum WCFS1 wild-type and mutant strains, PBMCs from 3 different donors were examined using 4 independent replicate wild-type and mutant L. plantarum cultures harvested during exponential-phase and stationary-phase of growth. Following 24 hr incubation at 37°C in 5% CO2, culture supernatants were collected and stored at -20°C until cytokine analysis. This time point was selected for analysis because previous studies showed that IL-12 levels remain unaltered after 4 days of L. plantarum incubation with PBMCs. Although IL-10 was shown to increase 2- fold after 4 days of co-incubation with L.