Millex hv pvdf

Lentiviral particles for shNTC, shHuR, shAgo2, miR-scr, miR-26a, eGFP-Stop and eGFP-miR-26-sponge were generated from HEK-293 T cells co-transfected with psPAX2, pcDNA3.1-VSV-G and the respective pFu3a plasmids using calcium phosphate coprecipitation. After 48 h virus production, supernatants were filtered (0.45 µm PVDF Millex-HV; Millipore), concentrated by ultracentrifugation (65,000xg, 140 min, SW 32 Ti rotor; Beckman Coulter) and resuspended in Opti-MEM™ (Life Technologies) [12 (link)].

pH measurements were carried out using Mettler Toledo Seven Easy pH meter (model S20) which was routinely calibrated with standard buffers. The experiments were performed at the 20 mM pH 7.4 sodium phosphate buffer. All preparations used in the experiments were filtered through 0.45 μm Millipore Millex-HV PVDF filter.

We used 7.5 ml of saline for washing each nasal cavity, which totalled 15 ml of saline per person. The collected fluid samples were combined and centrifuged at 500g x to separate the cells for cell counting and cell type analysis. The supernatant was centrifuged for the second time at 4000g x, before it was filtered through the 0.45 μm membrane (Millex-hv PVDF, Millipore) and divided into aliquots. Samples were frozen to -70°C for further use. Protein concentrations (RC DC Protein Assay, BioRad) of the NFL samples varied between 0.03 and 0.19 mg/ml. A total of 7–9 ml of each NLF sample was concentrated with ultrafiltration (Amicon Ultra-15 5000 MWCO, Millipore, Ireland) to 250 μl which was used in the proteomic analysis. For immunoblotting, 12 μl of untreated NFL was loaded onto the gel lane and for ELISA 250 μl was evaporated in SpeedVac to 50 μl.

The HIV-1 vector and plasmid used in this study have been described previously^{59 (link)}. HIV-eGFP virus (in which nef was replaced with the eGFP reporter gene) was produced by cotransfection with a ratio of 1:7 of pMD2.G together with pNL4–3_GFP_R-Env-^{60 (link)} (both plasmids were obtained from the NIH AIDS Research and Reference Program, Division of AIDS, NIAID, NIH, Bethesda, Maryland, USA). Vector containing supernatants were harvested 48 h after transfection in 293 T cells, cleared by centrifugation, filtered by a 0.45 mm filter (MILLEX-HV PVDF; Millipore, Carrigtwohill, County Cork, Ireland), and stored at −80 °C.
CTR, M1-MDM and M1² MDM were infected with 60 µl/well of HIV-eGFP viral stock. Infected MDM (5 × 10⁵ cells/condition) were detached from plastic adhesion after incubation with Accutase (150 µl/well for 30 min at 37 °C), as described^{46 (link)}, spun, and their pellet was resuspended in a fixing solution containing 4% paraformaldehyde (PFA). Flow cytometry for GFP expression was performed using a FACS Calibur instrument (Becton Dickinson Italia, SpA, Milano, Italy), and the results were analyzed with the FlowJo software version 8.4.3 (Tree Star, Ashland, Oregon, USA).