Cryopreservation and resuscitation
After cryopreservation, the seed cells can be preserved for easy access; reduce the risk of microbial contamination; reduce the risk of cross-contamination between cells; reduce genetic variation and morphological changes caused by subculture; avoid finite cells There is aging or malignant transformation. Cells should be identified and checked for contamination before cryopreservation.
The basic principle of cell cryopreservation and resuscitation is "slow freezing and rapid melting", which can preserve cell viability to the utmost. At present, cells are cryopreserved with glycerol or dimethyl sulfoxide (DMSO) as a protective agent. These two substances can improve the permeability of the cell membrane to water; slow freezing can cause the water inside the cells to ooze out of the cell and reduce intracellular ice crystals. Formation, thereby reducing the physical damage of ice crystals to cells. Resuscitation cells use a rapid melting method to ensure that extracellular crystallization melts in a short period of time, avoiding the infiltration of water into cells to form intracellular recrystallization due to slow melting. It is important to note that large amounts of heat are released when DMSO is diluted, so DMSO cannot be added directly to the cell fluid and must be prepared in advance.
- Cell cryopreservation
- 12-24 hours before cryopreservation, fresh medium was changed to keep the cells in logarithmic growth phase.
- The cells were trypsinized when the cells were as long as 80% confluent, and then boiled with fresh medium to prepare a cell suspension, which was centrifuged at 1000 rpm for 10 min. The suspended cells are directly centrifuged.
- Discard the supernatant, resuspend the cell pellet by adding the cryopreservation solution, and adjust the cell concentration to 3×10^6-1×10^7 cells/mL. The cell suspension was divided into frozen tubes, 1-1.5 mL per tube, and the lid was tightened. Mark the wall of the tube, indicating the name of the cell, the date of freezing, and so on.
- The temperature was sequentially lowered in the following order: room temperature → 4 ° C 20 min → -20 ° C 30 min → -80 ° C overnight → liquid nitrogen storage. It is also convenient to use the program cooling box. The cell cryotube can be placed in the program cooling box and placed directly at -80 °C overnight, and then transferred to the liquid nitrogen tank. Note that the amount of isopropyl alcohol in the program cooling box must be higher than the lowest scale line; after 5 times of freezing, isopropyl alcohol needs to be replaced once to avoid affecting the freezing effect.
- After cryopreservation of the cells, a tube of resuscitation should be taken to measure the survival rate of the cells. In theory, the cells can be stored for a long time in liquid nitrogen. For the sake of safety, the cells can be resuscitated after half a year of cryopreservation, and the growth of the cells is observed, and then frozen.
- Cell recovery
Cryopreserved cells are very fragile and must be handled gently. Except for a few cells that are sensitive to cryoprotectants (DMSO or glycerol), most cells can be directly inoculated into complete medium after thawing, and fresh medium is replaced every other day to remove DMSO and dead cells, thus avoiding large The problem that the cells cannot grow or adhere to the wall after partial thawing can also reduce the operation steps and reduce the probability of contamination because it is not centrifuged.
- The frozen cells were taken out from the liquid nitrogen tank, quickly thawed in a 37 ° C water bath, and the frozen tube was gently shaken to allow the cells to completely thaw within 1-2 min, passing through the most vulnerable temperature zone as soon as possible ( -5-0 ° C). Note that the tube of the frozen tube should not be submerged in water to avoid contamination.
- The frozen storage tube was wiped with 75% alcohol, placed in a clean bench, and the cells in the tube were transferred to a centrifuge tube containing the complete medium to prepare a cell suspension. Live cell counts were performed to adjust the cell concentration to at least 3 x 10^5 viable cells/mL.
- Transfer the cell suspension to a T25 cell bottle, add an appropriate amount of medium, gently shake the cell bottle to evenly distribute the cells, and incubate them in a thermostat.
- On the next day, cell adherent growth was observed and fresh medium was exchanged to remove DMSO and dead cells. Continue to culture until the cells are 80 to 90% confluent and normal passage. Generally, cells that have just recovered need to pass through 2-3 passages, and subsequent experiments can be performed after the cell viability is restored.
SARMs Capsules is a new performance-enhancing compound that is shaking up the fitness industry. They have the ability to promote muscle growth, fat loss, and even cardiovascular endurance. To maintain legitimacy, these new compounds sold for "research purposes" are emerging as promising alternatives to anabolic Steroids. A lot of people are there to promote a huge change in the body structure of SARM.
As we age, our endurance, strength, and skeletal muscle mass decline due to the gradual loss of type 2 muscle fibers. This severely limits our individual athletic performance. In this part of the androgen deficient population, the use of SARM, skeletal muscle mass, muscle strength will be significantly improved..
best sarms supplements,sarms capsules for sale, sarms tablets price,sarms pills for sale,buy sarms capsules
Shaanxi YXchuang Biotechnology Co., Ltd , https://www.peptidenootropics.com