3T3-L1 (mouse embryonic fibroblast)

Cell preservation method:

（1） Cell cryopreservation

1. Prepare a frozen culture medium containing 10% DMSO or glycerol and 10-20% calf serum;

2. Take cells in logarithmic growth phase, remove old culture medium, and wash with PBS.

3. Remove PBS and add an appropriate amount (covering the surface of the culture dish) to digest the monolayer grown cells;

4. Centrifuge at 1000rpm for 5 minutes;

5. Remove, add an appropriate amount of prepared frozen culture medium, gently blow and beat the cells evenly with a pipette, count, and adjust the final density of cells in the frozen culture medium to 5 × 106/ml to 1 × 107/ml;

6. Divide the cells into cryovials, with 1-1.5 ml per vial;

7. Mark the cell name, freezing time, and operator on the cryovial;

8. Freezing: The standard freezing procedure is a cooling rate of -1 to -2 ℃/min; When the temperature reaches below -25 ℃, it can be increased to -5 ℃ to -10 ℃/min; At -100 ℃, it can be quickly immersed in liquid nitrogen. Alternatively, the cryovial containing cells can be placed in a -20 ℃ freezer for 2 hours, then placed overnight in a -70 ℃ freezer, removed from the cryovial, and transferred into a liquid nitrogen container.

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Operation steps:

1) 24-48 hours before freezing, replace half or all of the culture medium to keep the cells in exponential growth phase.

2) Prepare a cryopreservation solution (prepared before use): Take another centrifuge tube, add culture medium and serum, and add dimethyl sulfoxide (DMSO) dropwise to a concentration of 20% to make a double cryopreservation solution. Place it at room temperature for later use.

3) Collect cultured cells by centrifugation, resuspend the cells in serum medium, and take a small amount of cell suspension (about 0 1 ml) Count cell concentration and pre freezing survival rate.

4) Take an equal amount of cryopreservation solution as the cell suspension, slowly add the cell suspension dropwise, and shake the test tube to prepare a cell cryopreservation suspension (DMSO concentration of 5-10%), with a cell concentration of 1-5 × 106 cells/ml. Mix evenly and divide into labeled cryopreservation tubes at 1-2 ml/vial. Take a small amount of cell suspension for contamination detection. After sealing tightly, indicate the cell name, generation, and date. Then freeze it.

Notes:

1) Cells intended for cryopreservation should be in a state of good growth and high survival rate, with a density of approximately 80-90%. Before freezing, check whether the cells still retain their properties. One to two days before freezing, test for the production of antibodies.

2) Cells can be frozen in liquid nitrogen for a long time without affecting cell viability; It can be stored for several months at -70 degrees Celsius.

3) Pay attention to the quality of cryoprotectants. DMSO should be of reagent grade, sterile and colorless (with a concentration of 0 Filter with 22micron FGLP Telflon or purchase sterile products directly, such as Sigma D-2650, in small volumes of 5-10 ml, store at 4 ℃ in the dark, and do not thaw multiple times. Glycerol should also be of reagent grade grade and stored in the dark after high-pressure steam sterilization. Use within one year after opening, as long-term storage may be toxic to cells. In this method, double freezing solution is prepared first to avoid damage to cells caused by the heat released when DMSO is directly added. Slowly adding cell suspension dropwise can gradually adapt cells to high osmotic pressure and reduce cell damage. DMSO may cause differentiation of some leukemia cell lines and can be replaced with 10% glycerol cryopreservation.

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