YAC-1 cell specific culture medium

Operation points:

Cell culture steps:

1、 Preparation of culture medium and culture cryopreservation conditions:

1) Prepare DMEM-H medium (with NaHCO3 1.5g/L added), 90%; Fetal bovine serum, 10%. Suspension culture medium can also be selected according to experimental needs to allow 293T cells to grow in suspension.

2) Cultivation conditions: Gas phase: air, 95%; Carbon dioxide, 5%. Temperature: 37 degrees Celsius, humidity in the incubator is 70% -80%.

3) Cryogenic solution: 90% culture medium, 10% DMSO, ready to use and prepared. Liquid nitrogen storage.

2、 Cell processing:

1) Resuscitate cells: Quickly shake and thaw a cryovial containing 1mL of cell suspension in a 37 ℃ water bath, then add 4mL of culture medium and mix well. Centrifuge at 1000RPM for 4 minutes, discard the supernatant, add 1-2mL of culture medium, and blow evenly. Then add all cell suspensions to the culture bottle and culture overnight (or add the cell suspensions to a 10cm dish, add about 8ml of culture medium, and culture overnight). The next day, change the solution and check the cell density.

2) Cell passage: If the cell density reaches 80% -90%, passage culture can be carried out.

For adherent cells, the following methods can be used for passaging:

1. Discard the culture supernatant and rinse the cells 1-2 times with PBS that does not contain calcium or magnesium ions.

2. Add 2 ml of digestion solution (0.25% Trypsin 0.53mM EDTA) to a culture bottle and place it in a 37 ℃ incubator for digestion for 1-2 minutes. Then observe the cell digestion under a microscope. If most of the cells become round and fall off, quickly take them back to the workstation, tap the culture bottle a few times, and add a small amount of culture medium to terminate digestion.

3. Add 6-8ml/bottle of culture medium, gently mix and aspirate, centrifuge at 1000RPM for 4 minutes, discard the supernatant, add 1-2mL of culture medium and blow evenly.

4. Divide the cell suspension into new dishes or bottles containing 8ml of culture medium in a ratio of 1:2 to 1:5.

3) Cell cryopreservation: When the cells are in good growth condition, cell cryopreservation can be performed. When freezing adherent cells, discard the culture medium and add a small amount. After the cells become round and fall off, add about 1ml of serum containing culture medium to the freezing tube, and then add 10% DMSO for freezing.

How to freeze and preserve cells?

Freezing preservation method 1: Place the freezing tube at 4 ℃ for 30-60 minutes → (-20 ℃ for 30 minutes *) → -80 ℃ for 16-18 hours (or overnight) → store in a liquid nitrogen tank vapor phase for long-term storage.

Freezing preservation method 2: Place the freezing tube in a programmable cooling machine that has been programmed to cool down 1-3 ℃ to below -80 ℃ per minute, and then store it in a liquid nitrogen tank vapor phase for long-term storage. -At 20 ℃, it should not exceed 1 hour to prevent excessive ice crystals from causing massive cell death. This step can also be skipped and placed directly in a -80 ℃ freezer, but the survival rate is slightly reduced.