2% NaCl followed by a hypertonic rescue in 1.5% NaCl. Finally, immune cells were fractioned by density

gradient centrifugation using Lympholyte Mammal (Cedarlane, Corby, Canada) and the mononuclear cell suspension containing a mixed population of T, B and antigen presenting cells (APCs) was suspended in complete DMEM supplemented with 10% FCS, Selleckchem LY3023414 50 μg/ml penicillin/streptomycin and 50 μg/ml gentamycin (Nacalai Tesque, Kyoto, Japan) [22, 23]. APCs (macrophages and DCs) were separated by their ability to adhere to glass as described before [21]. Briefly, cell suspensions (5 × 107 cells/well) were placed onto 2-well glass plates (Iwaki, Tokyo, Japan) and incubated for 2 h at 37°C and 5% CO2 to allow cells to adhere to the glass surface. Subsequently, they were washed gently with complete RPMI 1640 medium (Sigma) to remove non-adherent cells. With this methodology a mix population containing CD172a+mTOR inhibitor CD11R1−, CD172a−CD11R1low and

CD172a+CD11R1high cells was obtained [21]. Immunomodulatory effect of lactobacilli Evaluation of the immunomodulatory activity of L. rhamnosus CRL1505 and L. rhamnosus CRL1506 was performed using PIE cells and PPs-derived adherent cells [21–23]. For immunomodulatory assays, 1.5 × 104 PIE cells/well were plated onto type I collagen coated 24-well plates (Iwaki, Tokyo, Japan). Three days later, cell monolayers were washed, added with lactobacilli (5 × 108 cells/well) and incubated for 48 h at OSI-027 cell line 37°C and 5% CO2, after which cells were vigorously washed and harvested for total RNA isolation for cytokine expression profiles. In a second experiment to study immunomodulation of antiviral innate responses with lactobacilli, Celastrol PIE cell monolayers were incubated 48 h with lactobacilli, washed three times to eliminate possible stimulants and were further stimulated with poly(I:C) to mimic

viral infection at the indicated times. Again, RNA was isolated for studying expression profiles [22, 23]. Adherent cells were plated at a density of 1.5 × 106 cells/well in 12-well type I collagen-coated plates (Iwaki) or in 2-well glass plates (Iwaki). Lactobacilli were added to each well (5 × 108 cells/ml) and incubated for further 16 h. For evaluation of the modulation of antiviral responses by lactobacilli in APCs, adherent cells were prepared as indicated before and 16 h later, each well was washed vigorously with medium at least 3 times to eliminate bacteria; and finally the porcine cells were stimulated with poly(I:C) for the time indicated [21]. In addition, unlabelled anti-TLR2 rabbit IgG or anti-TLR9 rabbit IgG (Santa Cruz, Santa Cruz, CA) were used in blocking experiments. Cultured cells were incubated with the unlabelled anti-TLR2 or anti-TLR9 antibodies for 12 h before stimulation with lactobacilli. Lactobacilli immunomodulatory activity in PIE-adherent cells co-culture system Porcine PPs adherent cells suspensions were prepared as described above.