Cryopreservation and recovery of cells
1. Principle When the cells are directly frozen without adding any conditions, the water in the cells and the external environment will form ice crystals, which can cause mechanical damage, electrolyte increase, osmotic pressure change, dehydration,
PH changes, protein denaturation, etc., can cause cell death. If a protective agent is added to the culture medium, the freezing point can be lowered. Under slow freezing conditions, it can make the water in the cells permeate the cells before freezing. Storage at temperatures below -130 ° C can reduce the formation of ice crystals.
When the cell recovers, it should be fast, so that it can quickly pass through the most vulnerable cell-5 ~ 0 ℃, the cell can still grow, and the vitality is not damaged.
At present, the commonly used protective agents are dimethyl sulfoxide (DMSO) and glycerin, which are non-toxic to cells, have a small molecular weight, high solubility, and easily penetrate cells.
2. Operation steps (1) Frozen storage
1. Digest the cells (same as Experiment 2), and collect the cell suspension into a centrifuge tube.
2. Centrifuge at 1000 rpm for 10 minutes and discard the supernatant.
3. Precipitate and culture with protection solution, count, and adjust to about 5 × 106 / ml.
4. Divide the suspension into cryovials, 1 ml per tube.
5. Seal the cryopreservation tube tightly. If the ampoule is used, the flame is sealed, and the sealing must be strict, otherwise it will easily burst during recovery.
6. Put on a label to indicate the type of cells and the date of freezing. A metal heavy object and a string are tied to the cryotube.
7. Reduce the temperature in the following order: room temperature → 4 ° C (20 minutes) → refrigerator freezer (30 minutes) → low temperature refrigerator (-30 ° C for 1 hour) → gaseous nitrogen (30 minutes) → liquid nitrogen.
Note: Care should be taken during operation to avoid liquid nitrogen frostbite. Liquid nitrogen is regularly checked and replenished at any time. It must not be volatile and clean. Generally 30 litres of liquid nitrogen can be used for 1 to 1.5 months.
(2) Recovery
1. Prepare a tea pot or 1000ml burned out, containing 2/3 cup of 37 ° C warm water.
2. Remove the cryotube from the liquid nitrogen, quickly place it in warm water and keep stirring. Allow the frozen material in the cryotube to melt within 1 minute.
3. Open the cryotube and suck the cell suspension into the centrifuge tube.
4. Centrifuge at 1000 rpm for 10 minutes and discard the supernatant.
5. Add 10ml of culture solution to the pellet, pipette evenly, centrifuge again for 10 minutes, and discard the supernatant.
6. After adding the appropriate medium, transfer the cells to a culture flask and culture at 37 ° C. Observe the growth the next day.
3. Reagents and equipment: liquid nitrogen tank, cryopreservation tube (special cryopreservation tube or ampoule for plastic screw), centrifuge tube, pipette, centrifuge and other reagents: 0.25% pancreatin, culture medium, and culture with protective agent Base (ie cryopreservation solution)
Attachment: Preparation of cryopreservation solution:
Add glycerin or DSMO to the culture medium to make the final concentration reach 5-20%. The type and amount of protective agent varies with different cells. After preparation, store at 4 ℃.
Lipstick is a cosmetic product containing pigments, oils, waxes, and emollients that apply color, texture, and protection to the lips.
Many colors and types of lipstick exist. Some lipsticks are also lip balms, to add color and hydration.
Although the name originally applied to the baton (stick) of material, within a tubular container, usually around 10mm in diameter and 50mm in length the term now generally relates to the material itself, regardless of method of application.
Lip Stick,Makeup Powder,Matte Liquid Lipstick,Glimmer Liquid Lipstick
HENAN BON INDUSTRIAL(COSMETIC) CO.,LTD , https://www.boncosmetic.com