1. Matters needing attention:
1.1. The cells to be cryopreserved should be in a state of good growth (log phase) and high survival rate, about 80-90% density.
1.2. Check whether the cells still retain their unique properties before freezing. For example, hybridoma should be tested for antibody production one to two days before cryopreservation.
1.3. Pay attention to the quality of cryoprotectant. DMSO should be reagent grade, sterile and colorless (filtered with 0.22 micron FGLP Telflon or directly purchase sterile products, such as Sigma D-2650), packed in small volumes of 5 ~ 10 ml, stored at 4 oC in the dark Do not thaw multiple times. Glycerol should also be reagent grade, stored in autoclave and protected from light. Use within one year after opening, because it will be toxic to cells after long-term storage.
1.4. Cell concentration for cryopreservation:
1.4.1. Normal human fibroblast: 1 ~ 3 x 106 cells / ml
1.4.2. Hybridoma: 1 ~ 3 x 106 cells / ml, the cell concentration should not be too high, some hybridoma will die after thawing for 24 hours because the freezing concentration is too high.
1.4.3. Adherent tumor lines: 5 ~ 7 x 106, depending on the cell type. Adenocarcinoma requires a higher concentration after thawing, while HeLa only needs 1-3 x 106 cells / ml.
1.4.4. Other suspensions: 5 ~ 10 x 106 cells / ml, human lymphocyte must be at least 5 x 106 cells / ml.
1.5. The concentration of cryoprotectant is 5 or 10% DMSO. If you are not sure about the freezing conditions of the cells, you should also make a backup culture while cryopreservation to prevent the failure of freezing.
1.6. Freezing method:
1.6.1. Traditional method: 4 oC 10 minutes ---> -20 oC 30 minutes ---> -80 oC 16-18 hours (or overnight) ---> liquid nitrogen tank vapor phase long-term storage.
1.6.2. Program cooling: Use an isothermal cooling machine at a rate of â€“1 to -3 oC / min from room temperature to â€“120 oC, and place it in the liquid nitrogen tank vapor phase for long-term storage. It is suitable for the preservation of suspension cells and hybridoma.
2.1. Well-grown cultured cells
2.2. Fresh medium
2.3. DMSO (Sigma D-2650)
2.4. Sterile plastic cryopreservation tubes (Nalgene 5000-0020)
2.5. 0.4% w / v trypan blue (GibcoBRL 15250-061)
2.6. Hemocytometer and cover glass
2.7. Constant speed cooling machine (KRYO 10 Series II)
3.1. Change half or full medium before the day before freezing and observe the growth of cells.
3.2. Preparation of cryopreservation solution (preparation before use): add DMSO to fresh medium, the final concentration is 5-10%, mix evenly, and put at room temperature until use.
3.3. According to the operation of cell subculture, collect the cultured cells and take a small amount of cell suspension (about 0.1 ml) to count the cell concentration and survival rate before freezing.
3.4. Centrifuge, remove the supernatant, add an appropriate amount of cryopreservation solution to make the cell concentration 1-5 x 106 cells / ml, mix evenly, and aliquot it in the marked cryopreservation tube. A small amount of cell suspension is used for contamination detection.
3.5. Cryopreservation method 1: Place the cryotube at 4 oC for 10 minutes â†’ -20 oC for 30 minutes â†’ -80 oC for 16-18 hours (or overnight) â†’ liquid nitrogen tank vapor phase for long-term storage.
3.6. Cryopreservation method 2: Place the cryotube in the constant temperature cooling machine with the set program, and then put it into the liquid nitrogen tank. The program is: program 7: HB CELL
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