Journal: Current Protocols

Article Title: Using the Sleeping Beauty (SB) Transposon to Generate Stable Cells Producing Enveloped Virus‐Like Particles (eVLPs) Pseudotyped with SARS‐CoV‐2 Proteins for Vaccination

doi: 10.1002/cpz1.575

Figure Lengend Snippet: eVLP Yields at Different Scales of Production

Article Snippet: Stable producer HEK293T cell lines expressing eVLPs of interest (see Basic Protocol ) Complete DMEM (see recipe; make fresh), 37°C 20% (w/v) sterile sucrose (Sigma, cat. no. S0389; in 1× PBS) Sterile 1× PBS (Gibco, Fisher Scientific, cat. no. 10‐010‐031) T175 flasks (Falcon® Tissue Culture Flasks, Corning, 175 cm, plug seal, VWR, cat. no. 29185‐258) 50‐ml conical centrifuge tubes (CellPro™ 50 ml Centrifuge Tube, Polypropylene, Conical bottom, Alkali Scientific, cat. no. CW5602, or equivalent) Benchtop refrigerated centrifuge (Eppendorf Centrifuge 5810R, Marshall Scientific, cat. no. EP‐5810R, or equivalent), 4°C 0.45‐µm membrane filters (Medical Millex‐HP Syringe Filter Unit, 0.45 µm, polyethersulfone, 33 mm, Millipore Sigma, cat. no. SLHPM33RS) Ultracentrifuge tubes (38.5 ml, Open‐Top Thinwall Polypropylene Tubes, 25 × 89 mm, Beckman Coulter, cat. no. 326823) Scale Ultracentrifuge (Beckman Coulter Optima XPN‐80, cat. no. A95765, or equivalent) with swinging‐bucket rotor, 4°C Additional reagents and equipment for Bradford assay (see Support Protocol) NOTE : All the procedures should be performed in a BSL‐2 biosafety cabinet.

Techniques:

Journal: Current Protocols

Article Title: Using the Sleeping Beauty (SB) Transposon to Generate Stable Cells Producing Enveloped Virus‐Like Particles (eVLPs) Pseudotyped with SARS‐CoV‐2 Proteins for Vaccination

doi: 10.1002/cpz1.575

Figure Lengend Snippet: Schematic diagram showing the steps to produce three different output levels of eVLPs. ( A ) Cells are seeded in six T175 tissue culture flasks until they are confluent. ( B ) Cells are seeded in three five‐layer tissue culture flasks and concentrated using a Centricon Plus‐70 centrifugal filter device. ( C ) Cell are seeded in a Cell Factory System (10 layers) and concentrated using a Vivaflow 50R filter operated through a Vivaflow peristaltic pump. ( D ) The supernatants containing eVLPs from the three different methods (A, B, or C) are purified and concentrated by ultracentrifugation through a 20% (w/v) sucrose cushion. After 2 hr of ultracentrifugation at 100,000 × g , the pellets of VLPs will be concentrated at the bottom of the tube.

Article Snippet: Stable producer HEK293T cell lines expressing eVLPs of interest (see Basic Protocol ) Complete DMEM (see recipe; make fresh), 37°C 20% (w/v) sterile sucrose (Sigma, cat. no. S0389; in 1× PBS) Sterile 1× PBS (Gibco, Fisher Scientific, cat. no. 10‐010‐031) T175 flasks (Falcon® Tissue Culture Flasks, Corning, 175 cm, plug seal, VWR, cat. no. 29185‐258) 50‐ml conical centrifuge tubes (CellPro™ 50 ml Centrifuge Tube, Polypropylene, Conical bottom, Alkali Scientific, cat. no. CW5602, or equivalent) Benchtop refrigerated centrifuge (Eppendorf Centrifuge 5810R, Marshall Scientific, cat. no. EP‐5810R, or equivalent), 4°C 0.45‐µm membrane filters (Medical Millex‐HP Syringe Filter Unit, 0.45 µm, polyethersulfone, 33 mm, Millipore Sigma, cat. no. SLHPM33RS) Ultracentrifuge tubes (38.5 ml, Open‐Top Thinwall Polypropylene Tubes, 25 × 89 mm, Beckman Coulter, cat. no. 326823) Scale Ultracentrifuge (Beckman Coulter Optima XPN‐80, cat. no. A95765, or equivalent) with swinging‐bucket rotor, 4°C Additional reagents and equipment for Bradford assay (see Support Protocol) NOTE : All the procedures should be performed in a BSL‐2 biosafety cabinet.

Techniques: Purification

Journal: STAR Protocols

Article Title: Protocol for in vitro skin fibrosis model to screen the biological effects of antifibrotic compounds

doi: 10.1016/j.xpro.2021.100387

Figure Lengend Snippet:

Article Snippet: T-175 flasks , Greiner bio-one , 660175.

Techniques: Concentration Assay, Recombinant, Modification, Saline, Radio Immunoprecipitation, Protease Inhibitor, Acid Assay, Bicinchoninic Acid Protein Assay, Picogreen Assay, Inverted Microscopy