The concentrations of TNF Alpha, CXCL12 and CCL2 were measured in cell supernatants by using ELISA kits in accordance with the manufacturer NA s protocols (TNF Alpha and CXCL12 from R&D systems; CCL2 from) on an ETIMax-3000 reader. Supernatants were collected 24 h after infection, spun at 10,000 rpm for 10 min to discard bacteria and cell debris, and stored at −80°C until assayed.

Primary crypts were cultured according to Sato et al. using reduced concentrations of murine recombinant-spondin1 (500ng/mL –& ) and varying concentrations of EGF. Organoid structures were imaged at day 6. Cell proliferation was measured by BrdU incorporation by incubation with 20 Mu M BrdU for 1 hour at 37°C before fixation.

Bio-Gel injection and selected by adherence, were tested for their production of pro-inflammatory (IL-6, IL-1 Beta and TNF-Alpha ) and anti-inflammatory (IL-10) cytokines in response to stimulation by LPS and IFN-Gamma, in the presence or not of graded concentrations of recombinant chemerin (from 10−12 to 10−6 M).

Isolated crypts were counted and embeded in matrigel ( 356231 growth factor reduced) that contains 1 Mu M Jagged (Ana-Spec) at 5–10 crypts/Mu l and cultured in a modified form of medium as described in. Briefly, MEM/F12 was supplemented by EGF 40 ng/ml, Noggin 200 ng/ml-spondin 500 ng/ml (&or ) , N-Acetyl-L-cysteine 1 Mu M and Y-27632 dihydrochloride monohydrate 20 ng/ml. cADPR, when indicated, was added to culture at 50 Mu M. 30–50 Mu l drops of matrigel with crypts were plated onto a flat bottom 48-well plate ( 3548) and allowed to solidify for 20 to 30 minutes in a 37°C C. 350 Mu l of crypt culture medium was then overlaid onto the matrigel, changed every other day, and maintained at 37°C in fully humidified chambers containing 6% CO2. Clonogenicity (colony-forming efficiency) was calculated by plating 50 to 400 crypts and assessing organoid formation 3 to 7 days after initiation…