0.45 μm filter

Cell pellets were resuspended in 50mM Tris-HCl pH 7.5, 500 mM NaCl, 30 mM imidazole, 0.2% v/v TWEEN20 and EDTA-free protease inhibitor tablets and lysed using a cell disruptor (Constant Systems Ltd) at 14,000 psi. Following lysis, 10–50 μL Benzonase was added, and incubated at room temperature for 15 mins. Cell debris was removed by centrifugation at 30,000g for 30–40 mins at 4°C. The supernatant was passed through 0.45 μm filters (Sartorius), with additional incubation with Benzonase if required, before loading onto a 5 mL His-Trap FF column pre-equilibrated with equilibration buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, and 30 mM imidazole). Bound proteins were eluted with equilibration buffer including 500 mM imidazole. The eluate was then loaded directly onto a Superdex S75 gel filtration column pre-equilibrated with 30 mM Tris-HCl pH 7.5, 200 mM NaCl, and 1 mM TCEP. Buffer was then exchanged for 50 mM HEPES pH 7.5 with 150 mM NaCl either by gel filtration following N-terminal tag cleavage, or by concentration and dilution.

Lentiviral vector particle production was performed as described previously [20 (link)]. Briefly, 293T cells were seeded in 10 cm dishes in 10 ml DMEM. Cells were cotransfected 16 hrs post seeding with the plasmids pRRLsinhCMV-GFP-pre (a lentiviral vector genome encoding GFP) or pCSII-Luc (a lentiviral vector genome encoding luciferase), pMDLg/pRRE, pRSVrev, and pHIT-G or pIRES2-eGFP-CHIKV E3-E1 using Lipofectamine 2000 (according to the manufacturer’s protocol; Life Technologies). After 24 hrs incubation, the medium was discarded and replaced with 5 ml of fresh DMEM. Another 24 hrs later, the supernatant containing the vector particles was harvested, sterile filtered with 0.45 μm filters (Sartorius, Göttingen, Germany), and frozen at -80°C.

Lentiviral vector particle production was performed as described previously [18 (link)]. Briefly, HEK 293T cells were seeded in 10 cm dishes in a volume of 10 ml DMEM. Cells were cotransfected 16 hours post seeding with the plasmids pCSII-Luc, pMDLg/pRRE, pRSVrev, and pHIT-G or pIRES2-eGFP-CHIKV E3–E1 using Lipofectamine 2000 (according to the manufacturer’s protocol; Life Technologies). After 24 hours incubation, the medium was discarded and replaced by 5 ml of fresh DMEM. Another 24 hours later, the supernatant containing the vector particles was harvested, sterile filtered with 0.45 μm filters (Sartorius, Göttingen, Germany), and frozen at -80°C.

Lentiviral particles were generated in HEK293T cells cultured in DMEM with L-glutamine 2 mM and 10% fetal bovine serum. Cells were transfected with 1.7 μg pMD2.G, 3.3 μg psPAX2 and 5 μg of lentiviral vector (MISSION® pLKO.1-puro GFP shRNA, Sigma; target sequence 5′-CGTGATGAGTTCCCAATGATT-3′) in jetPEI®. Supernatants were collected and filtered through 0.45 μm filters (Sartorius) 24 and 48 h later. Transduction of MEC-1 cells was performed with Polybrene (Sigma) at 4 μg/ml by centrifugation at 900×g at 32 °C for 70 min without break. Cells were selected in 2.5 μg/ml puromycin 24 h after infection.

293T cells were plated at 1.1 × 10⁷ in T150 flasks and incubated overnight at 37°C. Transfections were carried out according to the manufacturer's instructions for adherent cells using Lipofectamine 2000 (Invitrogen). 8 μg psPAX2 and 5 μg pCMV‐VSVG helper plasmids (kindly gifted by Dr J. van Tuyn, Beatson Institute, Glasgow) were transfected with 20 μg of each pLenti‐PURO construct. Viral supernatants were harvested after 48 and 72 h incubations at 37°C, filtered through 0.45 μm filters (Sartorius, Epsom, UK) and concentrated by ultra centifugation for 2 h at 166 880g. Supernatants were frozen in aliquots at −80°C. Hs68 fibroblasts were plated at 8 × 10⁵ in 10 cm dishes and incubated overnight before infection at a multiplicity of infection (MOI) of 1 for 12‐16 h in the presence of 4 μg/mL polybrene (Sigma). Selection was performed using 2 μg/mL puromycin for 4 days. Control uninfected Hs68 fibroblasts died under these selection conditions. Following selection Hs68s were removed from the culture dish, counted for viable cells and used for experimental analysis.

Cells were labeled with either PKH-26 or with green florescent protein (GFP). For PKH-26 labeling, cells were dissociated by trypsin EDTA and then centrifuged, washed and incubated with PKH-26 [40 ] according to the recommended protocol by Sigma (PKH26GL, Sigma). Positive labeling was verified using fluorescence microscopy just before transplantation. For GFP labeling, viruses were produced by transient co-transfection of three plasmids into 293T cells as described earlier [41 (link),42 (link)], with several modifications. Briefly, 2 × 10⁶ 293T cells were transfected using the TransIT-293 Transfection Reagent (Mirus) with a total of 20μg of plasmid DNA: 3.5μg of the envelope plasmid pMD.G harboring the gene encoding VSV-G, 6.5μg of the packaging plasmid pCMVΔR8.91, and 10μg of the transfer vector expressing eGFP. The medium was replaced 24 hours after transfection with the MSC medium. The medium, which contains the viral particles, was collected 48 and 72 hours after transfection and filtered through 0.45-μm filters (Sartorius, Goettingen, Germany). The efficacy of the transfection was evaluated by FACS analysis.

One ml BAL or 500ul plasma were centrifuged for 5min at 1200g. Volumes were adjusted to 1.5ml with PBS and filtered through 0.45μM filters (Sartorious). To remove cell-free DNA and RNA, the supernatant was treated with RNase A (Qiagen) and TURBO DNase (Ambion) to final concentrations of 0.77mg/ml and 20Units/ml, respectively, for 1h at 37°C, and deactivated by Protease (Qiagen) treatment for 30min at 37°C.