Language
细胞培养
z-bo
2020-07-20
747
On the way to cell culture, there are as many things that need to be noted. In the small plates, the cells are dotted, attracting countless heroes to compete on the southeast branch. As the saying goes, "Everything is difficult at the beginning." Even the two seemingly simple things of cell recovery and preservation can cause an extremely large shadow area in Wang's heart. Fortunately, Zhonghong Boyuan has this cell culture cheat, which can completely turn the bear child into your intimate little padded jacket.
In fact, the newly revived cells are just like newly born babies. They are very fragile and require careful care of your friends. Otherwise, the cells will be played to death by us. In order to prevent cells from hanging up unclearly, we must be well aware of the various habits of cells.
1. As the saying goes, what mountain to sing, and different cells use different culture media. At this time, you have to mention the "Four King Kong" in the culture medium-MEM, DMEM, 1640, and F-12, which are basically involved in the culture process of more than 90% of the types of cells.
MEM has very comprehensive capabilities and is known as the most widely used cell culture medium.
The concentration of DMEM is 2~4 times higher, and it can be divided into high sugar type (contains glucose 4500mg/L) and low sugar type (contains glucose 1000mg/L).
1640 has relatively simple nutrients, a little less sugar and glutathione than DMEM, and is often used for lymphocyte culture.
F12 is commonly used to support the growth of CHO, Hela and L-cells and the culture of primary rat hepatocytes and prostate epithelial cells. Take 1/2 of each of the two media, MEM and F12, to form the most common media for neurobiology.
2. The spatial density of cell growth is the key to cell culture. When cultivating cells, you should explore the growth space density that the cells like. Neither make the cells in the cell flask sluggish due to overcrowding; nor can the cell concentration be lower than 1~5×10^5 cells/mL and cause "lonely death". "(Because the healthy growth of cells requires connections between cells). Therefore, before cell seeding, the cell concentration must be adjusted by cell counting.
3. When the morphology of the cultured adherent cells is not good, when passaging, discard the old medium, add 3ml of new medium (with or without serum) and wash once, suck it off with a dropper, and add 3ml of medium, gently pipette along the bottom of the bottle, and then aspirate. At this time, digestion and pipetting are officially carried out.
Secondly, add the cell suspension under pipetting to the culture flask containing the new medium, place it in the incubator, and observe the adherence of the cells according to the time point (10min, 20min, 30min). Then quickly pour out the medium, add 3ml of new medium and wash it gently, then add complete medium to culture and observe the cell growth and morphology. Until the perfect shape of the cell is found.
4. As for the amount of medium to add to the culture flask, it is necessary for the experimenters to find out. For fast-growing cells, add a little less medium to the cells that are easy to grow, and the cell morphology will be better, but pay attention to the grasp of the time of changing the medium.
5. How to choose a culture flask. Generally, if the cells with slow growth rate want a more beautiful cell state, the effect of using plastic bottles will be better than that of glass bottles; the effect of growing fast cells in glass bottles will be better. Therefore, for the same type of cell, when its growth rate is slow (such as when the cells are just resuscitated or in primary culture), the plastic bottle will be better; and when it is growing vigorously, the glass bottle will be relatively better.
6. When cells are frozen, the lid must be tightened, otherwise water will leak during the resuscitation bath and cause cell contamination. Therefore, the original tube and lid should be selected for the cryopreservation tube (different brands and models of tubes may vary), and it is best to seal the lid with sealing film after tightening. And each cryopreservation tube must be marked with the name of the cell and the freezing time, and recorded in the book, so as to avoid the subsequent recovery of the cells, the wrong cells.
7. Strictly perform gradient cooling during cell freezing (-4℃→-20℃~-40℃→-80℃→liquid nitrogen). To know that the living environment of the two heavens will only make the delicate cells explode and die, remember: slow down! But if you are really in a hurry, you can use the cell cryopreservation box to freeze the cells, and then transfer them to liquid nitrogen as soon as possible. In addition, the liquid nitrogen reserve in the liquid nitrogen tank should be measured regularly to ensure that all cells are immersed below the liquid surface.
8. When the cells are thawed, the water bath time is too long (2 minutes has not melted) due to the thicker wall of the cryotube and heat insulation, the temperature of the water bath can be adjusted to about 40 ℃, which can shorten the thawing time.
9. Book an ultra-clean table in advance to reduce the waiting time for inserting in the ice box after resuscitation, which can avoid overcrowding in the cell room, and the use of the ultra-clean table is strained. After 1 hour of resuscitation, no new culture medium has been added. (High concentration of DMSO prevents the formation of ice crystals in the cell and protects the cells during freezing, but after resuscitation and thawing, soaking for too long will be toxic to the cells)
10. You must be patient when resuscitating cells, because some cells will not really recover until a week after resuscitation. Therefore, even though there is no movement of the cells after three or four days of resuscitation, don't discard all the cells easily. You have to wait patiently and make a decision after two weeks.
11. Some notes about DMSO:
(1) DMSO can quickly penetrate the cell membrane and enter the cell, lower the freezing point, delay the freezing process, increase the ion concentration in the cell, reduce the formation of ice crystals in the cell, and reduce the degree of cell damage.
(2) DMSO will generate heat during the dissolution process, it should be mixed with the culture medium before adding serum. At the same time, the cryopreservation solution should be prepared in advance. DMSO may be more toxic to the cells;
(3) When resuscitating, centrifuge to remove DMSO, and wash 1-2 times with fresh medium. Be careful not to centrifuge too fast.