1. Take out the vigorous and soft calluses with tweezers, put them in a triangular flask and crush them gently. Every 100ml triangular flask is filled with 10~ 15ml sterilized MS medium, and each flask is inoculated with 1~ 1.5g callus to ensure enough cells in the initial culture.
2. Put the inoculated triangle on the rotary shaker. Shake flask culture was carried out at 65438 000 rpm and 25 ~ 28℃.
3. After 6~ 10 days of culture, if the cell proliferation is obvious, you can add 10ml fresh medium to the culture bottle, and if necessary, divide the culture into two bottles with a big mouth straw to continue the culture. (If the cells don't proliferate obviously, it may be that the starting materials are not suitable, so we should consider re-inoculating callus during the vigorous proliferation period). The first subculture can be carried out.
4. Filtration of suspension culture: After subculturing for several generations according to the "3" method, the culture solution should be mainly composed of single cells and small cell clusters (no more than 20 cells). If it still contains effective large cell clusters, it can be filtered with a metal mesh screen with an appropriate aperture, and then the filtered floating cells in the county will continue to be cultured.
5. Cell computing. Take a certain volume of cell fluid, add twice the volume of 8% chromium trioxide (CrO3), and put it in a water bath at 700℃ for 65438 05 minutes. After cooling, the cell suspension was repeatedly blown with a pipette to fully disperse the cells. After mixing, take a drop of county liquid and put it on the blood cell counting plate for counting.
6. Making cell growth curve: In order to understand the growth dynamics of floating culture cells in the county, the following methods can be used to draw the growth curve:
(1) fresh weighing method
At different times of subculture, a certain volume of suspended cell culture was collected by centrifugation, and the fresh weight of cells was weighed. With fresh weight as the ordinate and culture time as the abscissa, the growth curve of fresh weight was drawn.
(2) Dry weighing method.
After weighing the fresh weight, the cells can be dried, and then the dry weight can be weighed. With dry weight as the ordinate and culture time as the abscissa, the regeneration curve of cell stem cells was drawn.
The above two methods require sampling once every two days, seven times, and each sample is repeated three times. During the whole experiment, no fresh culture solution was changed into the culture bottle.
7. Examination of cell viability. For beginners, it is often necessary to detect the proportion of living cells. At different stages of culture, you can take a drop of cell fluid and put it on the glass slide, and drop a drop of 0. Stained with 1% phenol saffron solution (prepared with culture medium) and observed under a microscope. Several living cells are uncolored, while dead cells are quickly dyed red. You can also use 0. When stained with 1% fluorescent diacetate solution, all living cells will show blue fluorescence under the induction of ultraviolet light, and experienced operators can judge the life and death of cells according to cell morphology and cytoplasmic circulation.
8. Identification of cell regeneration ability: In order to know whether the cells cultured in suspension still have regeneration ability, the cultured cells can be transferred to agar-solidified medium to form callus at the base again, and then the differentiation of plants can be induced on the differentiation medium.