Normal human breast epithelial cell culture

Normal human breast epithelial cell culture

1. Collect milk 2-7d after delivery. Scrub the breast with sterile water and squeeze the milk into a sterile container. After mixing the collected milk, it is diluted with RPMI1640 to facilitate centrifugation.
2. Centrifuge the diluted milk (600-1000g, 20min), and gently aspirate the supernatant. In order to avoid affecting the pelleted cells, a small amount of liquid should be left. Wash the cells 2-4 times with RPMI1640 containing 5% FBS until the supernatant is not turbid.
3. Suspend the cells with growth medium, and add 50μl of cell suspension to a 5cm petri dish containing 6ml of medium. Culture cells at 37°C and 5% CO2.
4. Change the medium after 3-5 days, and then change the medium twice a week. Clones appear in 6-8 days. At the beginning, there are mixed growth of macrophages, acting as feeder cells. Macrophages gradually disappeared as the number of clones increased.
5. Subculture. Cells can be subcultured when 80%-85% of the bottom wall is overgrown. Add TEGPED to 1.5ml of each 5cm culture dish, use at 37℃, incubate the cells for 5-15min. Then, suspend the cells.
6. After centrifugation (100g, 5min), suspend the cells in culture medium and dilute the cell suspension 3 times. Inoculate the cell suspension into a culture dish or a culture flask, 2ml for every 3 culture dishes or every 1 culture flask.

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