First, the experimental principle
Cell culture can be divided into primary culture and subculture. The tissue cells obtained directly from the body are first cultured for primary culture; when the primary cultured cells reach a certain density, they need to be recultured, that is, after the cells are dispersed, from a container at 1:2 or other ratio Transfer to another container or several containers to expand the culture. For subculture, the cumulative number of subcultures is the algebra of the cells.
The basic principle of cell cryopreservation and resuscitation is slow freezing and rapid melting, which has been shown to maximize cell viability. At present, chloroglycerin or DMSO is used as a protective agent for cell cryopreservation. These two substances can improve the permeability of the cell membrane to water, and the slow freezing can cause the water inside the cell to exude outside the cell, thereby reducing the formation of intracellular ice crystals. Reduce cell damage caused by ice crystal formation. Resuscitation cells should be rapidly melted to ensure that extracellular crystallization is melted in a short period of time, avoiding the infiltration of water into cells to form intracellular recrystallization due to slow melting.
Second, the experimental method
material:
Mouse, saline, 100ml sterilized beaker, 15ml centrifuge tube, culture dish, dropper, sterile tweezers, scissors, mesh, foam board, pin, alcohol lamp, culture flask, culture solution, PBS, 0.25% trypsin , clean bench, carbon dioxide incubator, inverted microscope, microscope, counting plate, centrifuge, constant temperature water bath, refrigerator (4 ° C, -20 ° C, -70 ° C), liquid nitrogen tank, cryotube, cryopreservation , waste tanks, etc.
Frozen storage:
1. Pipette the cell suspension after passage, centrifuge, remove the culture solution, add the frozen solution, and dispense the frozen tube (the number of cells in the cryotube is generally (5 ~ 10) × 106 / ml, 2ml frozen tube Usually put 1 ~ 1.5ml cells).
2. Freeze by step
o Cryopreservation method 1: The standard cryopreservation procedure is the cooling rate of -1 to -2 °C / min; when the temperature is below -25 °C, it can be increased to -5 to -10 °C / min; to -100 °C, It can be quickly immersed in liquid nitrogen.
o Cryopreservation method 2: The cryotube is placed in the programmed cooling machine of the programmed procedure to reduce the temperature by 1 to 3 ° C to -80 ° C per minute, and then store it in liquid nitrogen for a long time.
recovery:
1. Remove the cryotube, immediately put it into a 37 ° C water bath for quick thawing, gently shake the cryotube to melt it in 1 minute, and transfer it into the aseptic table.
2. Open the cryotube and pipette the cell suspension into the centrifuge tube.
Centrifuge at 1000 rpm for 10 minutes and discard the supernatant.
4. After adding the appropriate culture solution, the cells were transferred to a culture flask, cultured at 37 ° C, and the growth was observed the next day.
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