Cell freezing experiments
Cell freezing experiments
Cryopreservation experiments of cells refer to the protection of cells cultured in vitro by adding liquid nitrogen. In order to maintain their various biological properties and reduce the consumption of culture vessels, culture solution, etc.
Principle
The basic principle of the cell freezing experiment is to use the low temperature property of liquid nitrogen and the two non-toxic substances, dimethyl sulfoxide (DMSO) or glycerol, to enter into the cell, so that the freezing point of the cell will drop, and can increase the permeability of the cell membrane to water, together with the method of slow freezing, which can make the water in the cell gradually penetrate out of the cell.
Operation method
Frozen Cells Experiment (Frozen Cells Experiment)
Principle
The basic principle of the cell freezing experiment is to use the low temperature property of liquid nitrogen and the two non-toxic substances, dimethyl sulfoxide (DMSO) or glycerol, to enter into the cell, so that the freezing point of the cell will drop, and can increase the permeability of the cell membrane to water, together with the method of slow freezing, which can make the water in the cell gradually penetrate out of the cell.
Materials and Instruments
Equipment: Move The basic process of freezing experiments of cells can be divided into the following steps:A. Digestion of monolayers of cells in the logarithmic growth phase with 0.25% trypsin solution according to the passaging method.B. Collect the digested cells in a centrifuge tube and centrifuge at 800-1000 r/min for 5 min.C. Discard the supernatant, add appropriate amount of freezing solution, blow the cells with a pipette to make suspension, and adjust the cell density to 5 x 106-1 x 107 cells/ml.D. Dispense 1 ml of cell suspension into each cryotube, screw the cap of the cryotube tightly and seal it tightly with wax film.E. Label the cryopreservation tubes with the name of the cells, the time of freezing, and the name of the person who froze the cells.F. Place the cryopreservation tubes under the following conditions: 4 ℃, 1 h→- 20 ℃, 2 h→- 85 ℃, 2 h→liquid nitrogen. Caveat 1. The cells in logarithmic phase have strong proliferative ability and high survival rate after freezing, therefore, cells in this phase should be selected as much as possible to be frozen when cell freezing is carried out. 2. In order to ensure the quality of cryopreservation and the survival rate of resuscitated cells, the digestion time should be mastered when cryopreservation, excessive digestion will cause damage to the cells, and it is difficult for the cells to survive when resuscitated. In addition, when inoculating after recovery, the concentration of cells should not be too low, and it is better to control at 5x106～1 x 107It is better to control the concentration of cells at 5x10 6 ~ 1 x 10 7 cells/ml, so as to ensure the success of resuscitation. 3. The cap of the freezing tube should be tightly capped to avoid spillage of cells during recovery; for some cells with poor freezing tolerance, such as embryonic cells, special care should be taken when freezing, and a layer of cotton can be wrapped around the freezing tube to avoid damage to the cells during freezing. For more product details, please visit Aladdin Scientific website.
① Cultured adherent cells (70% to 80% fusion)
② Centrifuge tube
① Centrifuge tube ③ Pipette
④ Freezing tube
⑤ Centrifuge tube
⑥ Liquid nitrogen tank
⑦ Ultra-clean bench
Reagents: ① 0.5% trypsin
① 0.5% trypsin
② Freezing solution
Liquid nitrogen