Doc et bibliographie
1001 mots
5 pages
MATERIALS AND METHODSMice, parasites and cells.
All animal care and procedures were performed in accordance with institutional guidelines and European regulations. Experiments were conducted using AQP9-/- and their litternate control aged from 7 to 10 weeks. P. yoelii (265BY) P. berghei (ANKA strain) and P. falciparum (NF54 strain) sporozoites were obtained by dissection of infected Anopheles stephensi mosquito salivary glands.
Human hepatocarcinoma cell lines HepG2-A16-hCD81EGFP (Yalaoui et al 2008 plos pathogens), HepG2-CD81 (Bartosh et al 2003 JBC) and Huh-7 were cultured in DMEM (Invitrogen) supplemented with 10% FCS, 2 mM glutamine and antibiotics (Invitrogen, Gibco).
Primary mouse hepatocytes were isolated as previously described (Renia et al., 1990), seeded at a density of 4 x 104 cells per well in 96-well microplates and cultured at 37 °C in 5% CO2 in William’s E medium (Invitrogen, Gibco) supplemented with 2% penicillin-streptomycin, 1% sodium pyruvate, 1% L-glutamine, 1% insulin-transferrin-selenium, 1% non essentials amino acids and 10% fetal bovine serum (Invitrogen, Gibco).
Human liver fragments were collected following informed consent provided from patients undergoing a partial hepatectomy. The collection and use of these tissues were undertaken in accordance with French government ethical regulations. Primary human hepatocytes were isolated from healthy parts of the liver biopsies as previously described (Carraz et al., 2006), seeded at a density of 8 x 104 cells in 96-well microplates precoated with rat tail collagen I (Becton Dickinson, Le Pont de Claix, France) and cultured at 37 °C in 5% CO2, in the same culture medium as above, supplemented with 10–7 M dexamethasone (Sigma, Saint Quentin Fallavier, France). Mouse and human hepatocytes were cultured for 24 h before silencing with siRNA.
Small interfering RNA and lentiviral transduction.
We used small double stranded RNA oligonucleotides targeting human CD81 (5’- GCA CCA AGT GCA TCA