Hoechst 33342 (Sigma-Aldrich) was used to determine cell viability. Flow cytometric analysis Volasertib cancer was performed on LSRII (BD Biosciences), and the data was analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA). Evaluation of the anti-inflammatory properties of Lc in vitro The LPS-activated macrophage cell line (RAW 264.7; ATCC TIB-71) was cultivated in the presence of different concentrations of bacterial lysate, as previously described [11]. Briefly, the cells were cultured for 24 hour at 37��C and 5% CO2 in Dulbecco’s modified Eagle’s medium (Institute of Molecular Genetics AS CR, Prague, Czech Republic) containing 10% heat-inactivated fetal bovine serum (Biochrom AG), 4.5 g/l glucose, 1.5 g/l sodium bicarbonate, 4 mM glutamine (Institute of Molecular Genetics AS CR), 100,000 U/l penicillin and 100 mg/l streptomycin (both Sigma-Aldrich).

The cells were cultured together with Lc, lysate of L. plantarum (Lp) or sterile PBS in the presence or absence of LPS (Salmonella typhimurium, 1 mg/l, Sigma-Aldrich). After cultivation, the concentration of TNF-�� in the supernatant was measured with ELISA (Invitrogen). The nuclear proteins were extracted from stimulated RAW264.7 cells by a nuclear extract kit (Active Motif, Rixensart, Belgium) and used to quantify the DNA binding activity of p65 subunit using the TransAM NF-��B family transcription factor assay kit (Active Motif). In NF-��B assay, only the concentration with the strongest immunomodulatory properties of Lc was used, i.e. 10 pg/l. All assays were performed according to the manufacturer’s recommendation.

Evaluation of microbiota changes by pyrosequencing Stool samples from PBS or Lc-treated mice, on day 0, 28 (just before DSS administration) and 35 (the last day of experiment) were collected. Total DNA from these samples was then isolated with ZR Fecal DNA Kit? (Zymo Research Corp., Orange, CA) according to the manufacturer’s recommendation. DNA was subsequently gel-purified and PCR was performed in triplicate for each primer pair, and pooled to minimize random PCR bias. The reaction mixture contained 1 ��l of DNA (10 ng/��l), 1.5 mmol/l MgCl2, 0.2 mmol/l of dNTPs, 1�� PCR buffer and 1 U platinum TAQ DNA polymerase (Invitrogen) and 0.40 ��mol/l of forward modified primer consisting of 454 adaptor A (5��-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3��; Genome Sequencer FLX system), unique 10-base tag sequence (ATATCGCGAG, CGTGTCTCTA, CTCGCGTGTC, TAGTATCAGC, TCTCTATGCG) and universal broad-range bacterial primer 5��-AYTGGGYDTAAAGNG and 0.

40 ��mol/l of reverse primer consisting of adaptor B (5��-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3��) and universal primer TACNVGGGTATCTAATCC. PCR conditions were as follows: 1��: 95��C, 3 min; 35��: 94��C, 50 sec; 40��C, 30 sec; 72��C, 60 sec; 1��: 72��C, 5 min and final AV-951 hold at 4��C.