These patients had macroscopically

These patients had macroscopically selleck products and histologically normal intestines, and had been referred with symptoms of rectal bleeding or change in bowel habit. Biopsy specimens were collected in ice-chilled complete medium and cultured within 1 hour in complete medium [Dutch modified RPMI 1640 (Sigma-Aldrich, Dorset, UK) containing 100 u/mL penicillin/streptomycin, 2 mM L-glutamine, 50 ��g/mL gentamicine (Sigma-Aldrich) and 10% foetal calf serum (TCS cellworks, Buckingham, UK)] in 24 well culture dishes (1 biopsy/ml/well) for 24 hours (37��C, 5% CO2, high humidity). Negative control involved the culture and handling in parallel of complete medium alone. A second negative control involved the use of human skin samples, following informed consent, of two patients during abdominal closure after colorectal surgery on non-cancer, non-IBD patients who were not genetically pre-disposed to colorectal cancer.

Skin samples were collected in complete medium. Incubation with Dispase II (Sigma-Aldrich, St. Louis, USA) was used to separate the epidermal and dermal layers which were subsequently cultured for 24 hours (37��C, 5% CO2, high humidity). Media were centrifuged in all cases (1500 rpm, t=5��) and cell-free biopsy culture supernatants (SN) used to detect STp-containing proteins. Protein concentration of SN was measured using the bicinchonic acid (BCA) Protein Assay kit (Pierce, Rockford, IL, USA), and extracted in Laemmli buffer 5 min at 90��C. STp-containing proteins were detected by western-blotting. Briefly, 5 ��g of protein extract from cultured biopsies were electro-transferred to a PVDF membrane for 30 min at a constant intensity of 50 V.

STp-containing proteins where detected with the specific IgG fraction as a primary antibody (11000), and with a anti rabbit IgG (HRP conjugated) as secondary antibody (12000) (Sigma). Pre-immunization serum, as negative control, confirmed the specificity of the reaction since it did not detect any STp-containing protein. Purified STp fragment (1 ��g) was also detected with the same procedure using a Penta?His? HRP conjugate from Qiagen. The Pierce CN/DAB Substrate Kit (Pierce), including both chloronapthol and diaminobenzidine, was employed as colorimetric substrate for HRP in all cases. Dendritic Cells from Peripheral Blood Human peripheral blood was collected from healthy volunteers with no known autoimmune or inflammatory diseases, allergies or malignancies, following informed consent.

Peripheral blood mononuclear cells AV-951 (PBMC) were obtained by centrifugation over Ficoll-Paque Plus (Amersham Biosciences, Chalfont St. Giles, UK). Human blood enriched DC were obtained following NycoPrep? centrifugation of overnight cultured PBMC. This protocol has been characterised in detail in previous studies from our laboratory as a way to obtain fresh human blood enriched DC [14].

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