V. vermiformis (ATCC CDC19) were cultivated in Peptone Yeast Extract Glucose (PYG) medium supplemented with 0.14 mg/mL penicillin (Sigma-Aldrich, USA), 50 mg/mL gentamicin (Thermo Fisher Scientific, USA), and 2.5 mg/mL amphotericin (Bristol-Myers Squibb, New York, USA) at 32 °C. For Orpheovirus production and purification, ten T175 cm2 flasks (Thermo Fisher Scientific, USA) containing 20 × 106 cells in PYG medium were infected with Orpheovirus at a multiplicity of infection (M.O.I.) of 0.01 and incubated for 4 days at 32 °C. The lysate was centrifuged at 1200 x g to remove cell debris. Then, the supernatant was collected, added over a 40% sucrose (Merck, Germany) cushion and centrifuged at 36,000 x g for 1 h. The pellet was re-suspended in PBS and stored at − 20 °C. Three aliquots of the virus stock were titrated to the 50% end-point and calculated by the Reed-Muench method [7 (link), 8 (link)].
T175 cm2 flasks
Manufactured by Thermo Fisher Scientific
Sourced in United States
The T175 cm2 flasks are standard cell culture vessels used for the growth and maintenance of adherent cells. These flasks provide a surface area of 175 cm2 for cell attachment and proliferation. They are made of tissue culture-treated polystyrene and are designed for optimal cell growth conditions.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
Amphotericin, by Bristol-Myers Squibb (1 mentions)
Stempro msc sfm xenofree medium, by Thermo Fisher Scientific (1 mentions)
2 protocols using t175 cm2 flasks
1
Orpheovirus Production and Purification
2
Expansion of Human Umbilical Cord Mesenchymal Stem Cells
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The hUC-MSCs were resuspended at 5 × 105 cells/cm2 in T-175 cm2 flasks (Thermo Fisher Scientific, USA), and hUC-MSCs were cultured in FBS-free DMEM medium (StemPro MSC SFM Xeno Free medium, Life Technologies, Gaithersburg, MD) for six days. Upon reaching 80% confluence, the monolayer was washed and harvested using Trypsin–EDTA (Thermo Fisher Scientific, USA), and the cells were split 1:4 into a new T-175 flask. The cells routinely replaced the medium when 80% of the mating was reached, which took approximately three days. The cultured medium was collected with a 0.2 μm filter replaced each time with the medium and stored in a deep freezer at −80 °C. We named it FM, which was kept in the flask.
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