Lenti x gostix plus

The mouse HMGB1 gene was stably transduced to the knockout cells or wild-type cells using a Lentivirus system. Preparation of viral vector was as follows: 1 μg of a mouse HMGB1 lentiviral cDNA ORF clone, pLV-mHMGB1-GFPSpark (Sino Biological) and 1 μg 3rd Generation Packaging Mix (Applied Biological Materials) was transfected into 5 × 10⁵ 293 T cells (Takara Bio) using 6 μl TransIT-Lenti transfection reagent (Mirus Bio, LLC) at 37 °C for 48 h according to the manufacturer’s instruction. Recombinant virus was concentrated by Lenti-X Concentrator (Takara Bio), checked for its titer using the Lenti-X GoStix Plus (Takara Bio) and stored at − 80 °C until use. Lentiviral vector (3800 GoStix values compatible to 9.8 × 10⁴ infection units) was transduced into 4 × 10⁴ cells (MOI = 2.5) of knockout clones or WT cells with 8 μg/ml polybrene at 37 °C for 48 h. Then, the culture medium was changed to fresh medium not containing polybrene and the cells were diluted and put into a well of 96-well plate (0.2 cells/well) for single cell cloning. Expression of HMGB1-GFP fusion protein was confirmed by western blot analysis. After confirmation of HMGB1 expression, the cells were expanded and used for further study within two weeks of in vitro culture to avoid loss of HMGB1 expression.

AAV-CCL19 virus was packaged as described in a previous study. Briefly, the AAV-CCL19 core plasmid and two helper plasmids were cotransfected into 293T cells. The cells were harvested 48 h later and purified according to the AAVpro Purification Kit (TaKaRa; 6232) instructions. After virus purification, virus titers were measured using the AAVpro Titration Kit (for real-time PCR) Ver. 2 (TaKaRa, 6233). For lentivirus packaging, the constructed core plasmid and two helper plasmids were cotransfected into 293 cells. The supernatant was recovered after 48 h, and the lentivirus was concentrated using the Lenti-X Concentrator (TaKaRa, 631231). The concentrated lentivirus was titered with Lenti-X GoStix Plus (TaKaRa, 631280).

Lentiviruses were prepared using LeGO-iG2 vectors and the Lenti-X packaging system (Takara Bio) and titered with the Lenti-X GoStix Plus (Takara Bio). Cord blood samples were collected from term infants during vaginal or cesarean deliveries. Mononuclear cells were isolated by density-gradient centrifugation on Ficoll-Paque Plus. CD34⁺ cells were purified using the Indirect CD34 MicroBead Kit (Miltenyi Biotec) according to the manufacturer’s protocol and cultured in StemPro-34 SFM medium (Thermo Fisher Scientific) supplemented with 100 ng/mL of thrombopoietin, stem cell factor, and Fms-related tyrosine kinase 3 ligand. After pre-stimulation for 18 h at 37 °C, 2 × 10⁵ cells were seeded in non-tissue culture-treated six-well plates pre-coated with 50 μg/mL of retronectin and transduced with lentiviruses for 48 h at a multiplicity of infection of 20. Transduced cells (GFP-positive) were purified by fluorescence-activated cell sorting using the BD FACSAria Fusion cell sorter. About 1000 sorted cells were mixed with the MethoCult methylcellulose-based medium (H4434, STEMCELL Technologies) and colonies were enumerated after 14 days of culture.

HEK293FT cells were cultured to ~90% in 15-cm plates fed with 15 mL IMDM (Caisson Labs) + 10% FBS (HyClone), 1% penicillin/streptomycin, and 2 mM caffeine. Cells were transfected with 10 ug transfer plasmid, 6 ug psPAX2 (Addgene #12260), and 4 ug pMD2.G (Addgene #12259) using LipoD293 (SignaGen) following the manufacturer’s instructions. The following day, medium was exchanged for IMDM + 2% FBS, 1% penicillin/streptomycin, 2 mM caffeine. Eighteen hours later, viral supernatant was filtered with a 0.45-μm filter. Virus was concentrated by mixing in an appropriate volume Lenti-X concentrator (Takara) and incubating at 4˚C overnight. Virus was pelleted by centrifugation for 45 min at 1,500 x g at 4˚C. Viral pellets were resuspended in an appropriate volume of culture medium for the target cell line. Resuspended virus was supplemented with 1:1000 polybrene (Santa Cruz Biotechnology). When appropriate, relative virus concentration (GoStix Value [GV, arbitrary units]) was approximated using Lenti-X GoStix Plus (Takara) following the supplied instructions and associated iPhone application v2.0.7. Colon organoids were transduced with 300 GV lentivirus. HCT116 and HCT116 TP53^-/- cells were transduced with 1000 GV lentivirus.