Hello! I am having problems in the cultivation of brain microvessel cells. I isolated cells from adult rats. In cultivation, I used 4 μg / ml puromycin for 2 days. (1 image) I have 3 problems: During passage, a significant number of cells die (~ 30-40%). Cells change morphology (especially at low density (2 images)). I noticed that cells of a different type were present in the culture and I decided to use puromycin again, at the same concentration, this led to the death of a large number of cells and a change in the morphology of the remaining ones. (3 image)
Hi!
In our lab, we are using mouse brain tissue to culture BMECs. The initial plating density is crucial on pre-coated Collagen IV wells. The cells should be confluent after 5-6 days. We add 10 µg/mL of puromycin the day of plating. The medium is changed on day 1 with the same concentration (10 µg/mL) of puromycin. On day 2, the medium is changed again with a diluted concentration of 4 µg/mL, Thereafter, the medium is changed with a concentration of 4 µg/mL every 3-4 days. As you know, puromycin is an inhibitor of protein synthesis. No puromycin is added after splitting cells. The cells don't appreciate passage, it's better to perform experiments directly w/o passages.
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