Can not know the whole strategy of cell culture _ cell subculture
1. When cells are grown to high density, they must be colonized into new culture flasks. The typical dilution ratio is 1:3 to 1:6, depending on the cell type.
2. Material:
2.1. Dulbecco's phosphate-buffered saline, Ca++/Mg++ free, D-PBS,
GibcoBRL 21600-010)
2.2. trypsin-EDTA solution (0.05% trypsin-0.53mM EDTA-4Na, GibcoBRL25300-062):
Store in 10 ml sterile 15 ml sterile tubes, store at –20 °C, and store in a 37 °C water bath before use.
2.3. Fresh medium
2.4. Sterile straws / centrifuge tubes / culture flasks
3. Steps:
3.1. adherent cells (adherent cells)
3.1.1. Aspirate the old culture solution.
3.1.2. Wash cells one or two times with D-PBS.
3.1.3. Add trypsin-EDTA solution (1ml/25cm2, 2ml/75cm2), and apply for several minutes at 37 °C. Observe under inverted microscope. When the cells are to be separated and appear round granular, trypsin-EDTA solution is aspirated. (If trypsin-EDTA is not removed, trypsin-EDTA is added, and the appropriate amount of serum-containing fresh medium is added to terminate trypsin. After centrifugation, the supernatant is aspirated.)
3.1.4. Tap the culture flask to remove the cells from the bottle wall, add appropriate amount of fresh medium, and pipette up and down several times to break up the cell mass. After mixing evenly, transfer to a new culture bottle according to the dilution ratio. , cultured under normal culture conditions.
3.2. Suspension cells
3.2.1. Aspirate the cell culture medium, place in a centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.
3.2.2. Aspirate the supernatant, add an appropriate amount of fresh medium, mix well, transfer to a new flask according to the dilution ratio, and culture under normal culture conditions.
3.3. Hybridoma (hybridoma)
3.3.1. Some hybridoma cells need to be cultured for more than three days to produce antibodies. If the medium is changed, antibodies may be lost. Therefore, subculture can be changed without changing the medium after centrifugation, and the cell concentration can be diluted by directly adding fresh medium. If the volume is too large, it can be placed obliquely or colonized into a new culture flask.
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