Host and parasite strains and protein sellekchem sample preparation Ethics statement Our laboratory has received the permit N�� A 66040 for experiments on animals from both French Minist��re de l’Agriculture et de la P��che and French Minist��re de l’Education Nationale de la Recherche et de la Technologie. Housing, breeding and animal care of the mice followed the ethical requirements of our country. The experimenter possesses the official certificate for animal experimentation delivered by both ministries (D��cret n�� 87�C848 du 19 octobre 1987; number of the authorization 007083). Parasite and host breeding and in-vitro culture procedures Two strains of S. mansoni were used in this study, a Brazilian strain and a Guadeloupean strain the first of which is compatible (C strain) and the second of which is incompatible (IC strain) with a single Brazilian mollusc strain [28].
Each strain was maintained in (i) their sympatric strain of B. glabrata and in (ii) hamsters (Mesocricetus auratus) as described previously [28]. Miracidia from S. mansoni C and IC were hatched from eggs axenically recovered from 60-days infected hamster livers, according to the previously described procedure [26]. Briefly, livers were collected and kept overnight at 4��C in sterile saline solution (NaCl 150 mM), containing an antibiotic/antimycotic mixture (penicillin 100 units/ml, streptomycin 0.1 mg/ml, amphotericin B 0.25 ��g/ml; Sigma). The livers were then homogenized and the eggs were filtered and washed. Miracidia were hatched from eggs in sterile water.
Miracidia were recovered by pipetting and concentrated by sedimentation on ice Dacomitinib for 1-h and directly submitted to in vitro transformation to obtain primary sporocysts (Sp1) [29]. Miracidia were cultured at 26��C in sterile Chernin’s balanced salt solution (CBSS, [30]) containing the antibiotic/antimycotic mixture previously described [31]. Full transformation of miracidia to Sp1 occurred within 24 hours. Sporocysts were spun down (600 g for 5 min) and frozen at ?80��C. Native extraction of sporocysts For each strain, 40,000 sporocysts were resuspended in 200��l TBS containing tween 20 (0.05%, v/v) and antiprotease cocktail (complete protease inhibitor cocktail, Roche). Then, they were submitted to sonication (Vibracell 75185 apparatus, 4 pulses of 20 seconds at 20% of amplitude on ice).
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