PDCs) and PBMCs depleted of pDCs (PBMCs-pDCs) had been isolated by adverse selection, along with the cells were resuspended in RPMI 1640 medium supplemented with two mM L-glutamine, 100 Units/mL penicillin, 100 /mL streptomycin and 10 heat-inactivated FBS. Since human key pDCs are present in a extremely low quantity in blood (0.2.five of PBMCs), to facilitate the biochemical analyses of cell signalling, a set of TLR4 Activator Species experiments have been carried out employing GEN2.two, a pDC cell line derived from a leukaemia patient [38], deposited within the CNCM (Collection Nationale de Cultures de Microorganismes, Pasteur Institute, Paris) on 24 September 2002, under the quantity I-2938. The pDC cell line was obtained by means of a signed Material Transfer Agreement (MTA). The proliferation of GEN2.two cells is strictly dependent on the presence of a feeder layer created by the murine stromal cell line MS-5 (deposited within the DSMZ (German Collection of Microorganisms and Cell Cultures) beneath the No. ACC441). GEN2.2 cells had been cultured in flasks precoated using a NPY Y5 receptor Antagonist Synonyms sub-confluent irradiated MS-5 monolayer in RPMI 1640 medium containing 1 glutamax (Gibco, Gibco, Amarillo, TX, USA, cat. 35050-038), 100 Units/mL penicillin, one hundred /mL streptomycin, 1 mM sodium pyruvate (cat. ECM0542D, Euroclone, Milan, Italy), 1 nonessential amino acids (Euroclone, Milan, Italy, cat. ECB3054D) (hereafter referred to as total medium) and 10 ultra-low endotoxin FBS (cat. S1860-500, Microtech, Naples, Italy), previously inactivated at 56 C for 30 min. GEN2.2 cells had been maintained in culture for no more than two months and, only the CD45+ non-adherent fraction, corresponding to GEN2.2 cells in the supernatant, was applied for the experiments. THP-1 cells (from American Kind Culture Collection, or ATCC), derived from a human monocytic leukaemia have been grown in RPMI 1640 medium supplemented with 2 mM Lglutamine, 100 Units/mL penicillin, 100 /mL streptomycin and 10 heat-inactivated ultra-low endotoxin FBS. For the experiments, THP-1 have been seeded at 100.000 cells/cm2 and added with 35 nM of PMA (phorbol 12-myristate 13-acetate) (cat. P8139, Sigma-Aldrich, Milan, Italy) to adhere and differentiate, acquiring a macrophage-like phenotype which mimics, in lots of respects, major human macrophages [39]. In certain, following a PMA therapy of 32 h, the medium was replaced with fresh medium supplemented with 20 FBS. Then, right after 1 day of resting, differentiated THP-1 had been applied for the experiments. All cells had been maintained at 37 C in an incubator having a 5 CO2 humidified atmosphere. two.2. Recombinant Nef Protein Preparations and Reagents Wild form recombinant myristoylated Nef protein and a mutant within the acidic cluster E66 EEE69 AAAA present at N-terminal end (referred to as myrNefSF2 w.t and myrNefSF2 4EA, respectively) were generated from HIV-1 SF2 allele in the laboratory of Dr. Matthias Geyer at Max-Plank-Institut fur molekulare Physiologie, Dortmund, Germany, as previously reported [40]. Briefly, these proteins had been obtained by co-transformation of anViruses 2022, 14,4 ofE. coli bacterial strain with two plasmidic expression vectors containing a codon-optimized Nef or perhaps a N-myristoyl-transferase coding sequence, respectively, and purified as C-terminal hexahistidine-tagged fusion proteins. The myristoylation of recombinant Nef proteins was verified by mass spectrometry at Dr. Geyer’s laboratory. All Nef preparations were analysed for the presence of endotoxin as a contaminant employing the chromogenic Limulus amebocyte lysa.