W2 strains were maintained in HYPERFlasks (Corning, Corning, NY, USA) in 500 mL RPMIc [RPMI 1640 media supplemented with 0.25 % Albumax II (GIBCO, Grand Island, NY, USA), 2 g/L sodium bicarbonate, 25 mM HEPES (pH 7.4), 0.1 mM hypoxanthine, and 50 ug/L gentamycin] in a 37 °C, 5 % O2, 5 % CO2 incubator in 2 % haematocrit (HC). Cells were synchronized with 5 % sorbitol treatment for two generations to achieve high synchronicity.
Hyperflask
Manufactured by Corning
Sourced in United States
Hyperflasks are cell culture vessels designed by Corning to provide a larger surface area for cell growth. They feature a unique three-dimensional structure that maximizes the available growth surface while maintaining a compact footprint. Hyperflasks are made of high-quality materials and are intended for use in various cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Tryple, by Thermo Fisher Scientific (3 mentions)
Albumax 2, by Thermo Fisher Scientific (2 mentions)
Pei 25 kda, by Polysciences (2 mentions)
P24 elisa, by Takara Bio (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Benzonase nuclease, by Merck Group (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Kta purifier, by GE Healthcare (2 mentions)
Vivaflow 200 cross filtration device, by Sartorius (2 mentions)
Superdex 200 increase column, by GE Healthcare (2 mentions)
Hek293 c18 cells, by American Type Culture Collection (2 mentions)
Amicon ultra 15, by Merck Group (2 mentions)
Prime xv, by Irvine Scientific (2 mentions)
Nucleocounter, by ChemoMetec (2 mentions)
Vivaflow 200, by Sartorius (1 mentions)
Peg 8000, by Hampton Research (1 mentions)
Exosome depleted fbs, by Thermo Fisher Scientific (1 mentions)
Pluronic f 68, by Thermo Fisher Scientific (1 mentions)
43 protocols using hyperflask
1
Maintaining Synchronized P. falciparum Cultures
2
Comparative Evaluation of AAV Vectors
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AAV1, AAV3B, AAV5, AAV9, AAVrh74, AAVDJ, and Spark100 vectors expressed Firefly luciferase (FLUC) from a ubiquitous promoter. Except Spark100, all vectors were obtained from the Viral Vector Core Facility at UMASS. Spark100 was produced at the Rare Disease Research Unit, Pfizer, Cambridge, MA following triple transfection of adherent HEK293 cells grown in HYPERFlasks (Corning). In brief, cell lysates and supernatant were clarified and tangential flow filtration concentrated before subjecting to Iodixanol gradient centrifugation. Isolated fractions were dialyzed, tittered, and stored at −80°C in PBS/5% glycerol before use.
3
Engineered rAAV2/2[MAX] Vector Production
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rAAV vectors were manufactured as per our previously published protocol.47 (link) Briefly, HEK293T cells were seeded into HyperFlasks (Corning, New York, NY, USA) and triple transfected using a 2:1 PEI-to-DNA ratio with (1) a pTR plasmid containing a red fluorescent reporter protein (mCherry) expressed ubiquitously from a CBA promoter flanked by AAV2 inverted terminal repeats (ITRs); (2) a helper plasmid containing adenoviral elements (pHelper); and (3) a plasmid encoding AAV2 Rep and Cap genes for a mutant capsid, termed rAAV2/2[MAX], that contains five previously elucidated point mutations (QuadYF-TV) and the 7m8 peptide insertion.10 (link),13 ,14 (link),48 (link) Three days post-transfection, cells were harvested, lysed, and purified via iodixanol ultra-centrifugation followed by buffer exchange using a 100 kDa centrifugation column (Amicon, Darmstadt, Germany) into HBSS containing 0.014% Tween 20.49 (link) The rAAV titer was determined with a PicoGreen assay as described previously.50 (link)
4
Isolation and Concentration of Exosomes
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F3 and F3.TSG cells were seeded in Hyperflasks (Corning, NY, USA) with 2.5 × 107 cells and cultured for 2 days in an incubator until 90–100% confluence. To obtain conditioned medium for exosome isolation, the cultures were washed 3 times using 300 mL PBS each time, and the medium was replaced with 500 mL of phenol red-free DMEM; the cells were subsequently cultured for 3 days in an incubator. Then, the conditioned media were filtered using a 0.2 μm filter top system (Corning) to remove cell debris [58 ]. Then, the filtered media were subjected to ultracentrifugation (120,000× g at 4 °C for 2 h) to collect exosome. The total exosomes were concentrated 10 times (v/v) using Vivaflow 200 (M.W. cutoff 100 kDa, Sartorius AG, Gottingen, Germany), and freeze-dried (LP-10; Ilshinbiobase Co., Ltd., Seoul, South Korea). The isolated exosomes were stored at −80 °C and resuspended in PBS for treatment.
5
Lentiviral Library Packaging Protocol
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The CHESS scFv synthetic library was packaged into replication-incompetent lentiviral particles in HEK-293T cells. 2 × 107 cells were seeded 3 days before transfection in hyperflasks (Corning, catalog no. 10020). The cells were transfected with 760 µg of total DNA (CHESS library and lentiviral packaging vectors) and 1900 µg of branched polyethyleneimine transfection reagent (Sigma–Aldrich, catalog no. 408727). The resulting lentiviral supernatants were harvested 3 days later, clarified by centrifugation at 800 × g for 10 min, and stored frozen at −80 °C. The physical titer of the lentiviral preparations was determined by quantitative PCR, and the infectious titer on CHO–EpCAM cells of lentiviruses encoding GFP was determined by flow cytometry. The infectious titer of the library lentiviral preparation was calculated by using the physical/infectious titer ratio determined for the GFP lentiviruses.
6
Large-scale expansion of hiHep cells
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After expansion in 100 mm dishes, final expansion of hiHep cells was performed in Hyperflasks (Corning). Finally, about 3 × 109 hiHep cells were harvested from the Hyperflasks and used for the hiHep-BAL treatment.
7
High-titer LSDV-based vaccine stock preparation
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High titer stocks of LSDV-SODis-p67HA-BLV-Gag and nLSDVSODis-UCT were prepared by infecting MDBK, LT or BHK-21 cells in 175 cm3 flasks or HYPERFlasks® (Corning®, USA) at MOIs 0.0025 to 0.005. Once all cells were infected and about 50% of cells had lifted, the flasks were frozen and thawed twice and lysates were clarified by low speed centrifugation at 320 x g for 10 min. Supernatants were placed into SS34 tubes and underlaid with 1-1.5 ml of 36% (w/v) sucrose diluted in 1X PBS. Viruses were pelleted by centrifugation at 27 000-39 000 x g for 1-2 hrs at 4°C. The pellets were resuspended in 1X PBS and stored at -80°C until needed. Prepared stocks were titrated by infecting MDBK cells in 96-well plates with serial dilutions of the stock (10-1 to 10-12 in DMEM) and tissue culture infectious dose at 50% infection (TCID50) was determined using the method described by Reed and Muench (37 (link)).
8
Quantifying HBV DNA Production
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HepAD38 cells were grown in DME/F12 medium containing 4% FBS and 1% dimethyl sulfoxide (DMSO) in HyperFlasks (Corning). Cell culture supernatants were collected every 6 days and were used for HBV concentration by precipitation with 10% polyethylene glycol (PEG) 8,000 (Hampton Research). The genome copy numbers of HBV DNA were determined by a real-time PCR method.
9
Production of AAV9 Vectors for shRNA Delivery
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AAV vectors were produced by the triple transfection method using HEK293T cells [31 (link),32 (link)]. The shuttle plasmids pAAV[shRNA]-EGFP-U6 > mZnf219[shRNA#1] or [shRNA scramble] (VectorBuilder) were packaged into AAV9 capsids using the helper plasmids pAdDF6 and plasmid pAAV9 (providing the rep and cap viral genes) (PennVector). All plasmids were co-transfected into HEK293T cells using linear polyethylenimine (MW 25,000). The cells were seeded in Hyperflasks (Corning) at 1.2 × 108 cells per flask the day before transfection.
Transfection and viral particle collection were performed as previously described [33 (link)]. Sequences of sh-RNAs are included inTable S4 .
Transfection and viral particle collection were performed as previously described [33 (link)]. Sequences of sh-RNAs are included in
10
Fibroblast Exosome Conditioning Protocol
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Hyperflasks (Corning) were used to grow cardiac fibroblasts in fibroblast culture media until at least 70% confluence was achieved. After washing with PBS, cell cultures were incubated for 48 h in exosome-depleted fibroblast maintenance media (DMEM, 2% v/v Exosome-depleted FBS (Gibco, Waltham, MA, USA), 1% v/v Penicillin-Streptomycin). Following conditioning of the media, the media was removed and filtered through a 0.45 µm bottle-top filter.
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