For the isolation of NSC-derived EVs, frozen vials containing passage 11 NSCs were thawed at 37°C and plated on to a T-75 culture flask (Corning) and grown at 37°C in a CO2 incubator. Following 70% confluency, the cells were dislodged using 1 U/ml of dispase (Gibco), washed with NSC media (Gibco), and seeded at ~500 cells per cm2 into 150 × 20 mm diameter tissue culture plates (Corning) in NSC expansion medium. Once NSCs reached 90% confluency, the media was harvested and used for isolating EVs or stored at −80°C for further use.
Nsc media
Manufactured by Thermo Fisher Scientific
NSC media is a serum-free, chemically defined medium designed for the culture of neural stem cells. It provides the necessary nutrients and growth factors to support the growth and differentiation of neural stem cells in vitro.
Automatically generated - may contain errors
2 protocols using nsc media
1
Isolation of NSC-Derived Extracellular Vesicles
2
Quantifying Glioblastoma and Glioma Cell Colony Formation
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Colony formation assays using U87 cells were carried out as follows: 300 cells were seeded onto 10 cm dishes and treated with TMZ for 2 h at 37°C in a humidified atmosphere of 5% CO2. The medium was then replaced and cells were allowed to form colonies for 15 days. Colonies were stained using crystal violet solution (0.1% Brilliant blue R (Sigma-Aldrich) in PBS) and quantified using ImageJ/FIJI analysis software.
For NCH644 cells, soft agar colony formation assays were carried out as follows: 6 × 103 NCH644 cells were suspended in 0.3% low melting agarose (LMA) containing 1 ml Neural Stem Cell media (NSC-media, Gibco) supplemented with TMZ or vehicle, and then seeded on top of pre-coated 0.6% LMA (1 ml) containing NSC-media in the six-well plate. Cells were incubated for 21 days at 37°C with twice-weekly fresh medium supplementation (200 μl). After 3 weeks the cells plates were stained with crystal violet for imaging with ImageQuant TL and colony quantification.
For NCH644 cells, soft agar colony formation assays were carried out as follows: 6 × 103 NCH644 cells were suspended in 0.3% low melting agarose (LMA) containing 1 ml Neural Stem Cell media (NSC-media, Gibco) supplemented with TMZ or vehicle, and then seeded on top of pre-coated 0.6% LMA (1 ml) containing NSC-media in the six-well plate. Cells were incubated for 21 days at 37°C with twice-weekly fresh medium supplementation (200 μl). After 3 weeks the cells plates were stained with crystal violet for imaging with ImageQuant TL and colony quantification.
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