Serum free cell cryopreservation solution

​ 无血清（Serum Free）细胞冻存液为即用型产品，不含血清和动物来源性蛋白。因此，不仅降低了各类细菌、病毒、支原体等污染风险，保证冻存细胞的安全，而且减少外源性蛋白对细胞正常生长和分化的影响。此外，无需程序降温，可长期-80℃保存细胞，适用于绝大多数哺乳动物细胞的冻存。 注意事项： 1. 使用前确保细胞冻存液彻底融化，并轻轻混匀。融化后的细胞冻存液置于4ºC保存，三个月内有效。 2. 本产品含有酚红指示剂，长时间敞口会导致PH变化，并出现沉淀。故使用过程中请尽量缩短敞口时间，使用完毕后盖紧盖子保存，如有必要可适当分装使用。若使用一段时间后出现少量沉淀，经测试对细胞冻存无影响，去除沉淀可采取离心取上清、0.22 μM孔径过滤器过滤等方法去除。 3. 细胞冻存前请确保其状态良好，活细胞比例宜大于90%。 4. 本产品含DMSO，部分对DMSO敏感的细胞，建议进行预实验。 5. 对于某些珍贵的细胞，建议同时使用含FBS的常规冻存液进行对比测试，确认性能后再进行正式冻存。 6. 冻存细胞分装后应尽快移入-80ºC超低温冰箱内，减少在外存放时间。 7. 为了您的安全和健康，请穿实验服并戴一次性手套操作。 使用方法： 1. 细胞冻存 （1）取出细胞冻存液，待完全融解并混匀后置于 4℃保存，待用。 （2）贴壁细胞：移除细胞培养基，用无菌 1×PBS 轻轻清洗细胞一次以除去残余的血清，再加入适量的胰酶消化液消化细胞。待消化完成后立即加入适量含血清的细胞培养基以终止胰酶，随后轻轻吹打细胞，并适当吹散和重悬。 注：不可过度消化细胞，以刚好能把细胞吹打下来为最佳。过度消化后的细胞由于生长状况较差，通常不宜再冷冻保存。吹打和重悬的过程也应轻柔，否则可能会影响细胞复苏时的存活率。 悬浮细胞：从步骤（3）开始执行。 （3）将细胞悬液转移至合适的离心管中。 （4）细胞计数，计算细胞总数和所需细胞冻存液的量。细胞的冻存密度一般为 1×106-1×107cells/mL。（6.7为上标） （5）100-200 xg，离心 5-10 min，弃上清。离心速度和时间取决于细胞类型。 （6）加入计算好的细胞冻存液体积，用移液枪轻轻吹打以重悬细胞，根据细胞类型调整细胞密度（一般为 1×106cells/mL 或更高）。（6为上标） （7）分装。将上述细胞悬液分装于 1.5 mL 或 2 mL 细胞冻存管中，并做好标记。 注：如果后续放入液氮罐中保存，须使用可用于液氮冻存的细胞冻存管。 （8）将细胞冻存管放置于-80ºC 冰箱中。 注：如果后续拟放入液氮中长期保存，可在-80ºC 冰箱内保存 24 h 后移入液氮罐内。 2. 细胞复苏 （1）从-80ºC 冰箱或液氮中取出冻存管，立即放入 37ºC 水浴锅中，轻轻晃动少于 1 min，直至残留小部分冰块。 （2）将上述细胞悬液转移至 15 mL 无菌离心管中，加入 5-10 mL 预热的完全培养基，轻轻混匀。 注：对于一些复苏效率很高的细胞也可直接将细胞悬液转移至离心管进行下一步。 （3）100-200 ×g，离心 5-10 min，离心速度和时间取决于细胞类型。 （4）沉淀细胞，小心去除上清。 （5）加入适量预热的完全培养基，轻轻吹匀后转移至培养器皿中，置于细胞培养箱中培养。 应用范围： 细胞冻存 Uelandy serum free cell crYOpreservation solution is a ready to use product without serum and animal derived protein. Therefore, it not only reduces the pollution risk of all kinds of bacteria, viruses, mycoplasma, etc., and ensures the safety of frozen cells, but also reduces the impact of exogenous proteins on the normal growth and differentiation of cells. In addition, without the need for programmed cooling, cells can be stored at -80 ℃ for a long time, which is suitable for the crYOpreservation of most mammalian cells. Matters needing attention： 1. before use, ensure that the cell cryopreservation solution is completely melted and gently mixed. The thawed cell cryopreservation solution is stored at 4 º C and is effective within three months. 2. this product contains phenol red indicator. Long time exposure will lead to pH change and precipitation. Therefore, please try to shorten the exposure time during use. After use, close the cover tightly and store it. If necessary, it can be properly subpackaged for use. If a small amount of precipitate appears after using for a period of time, it has no effect on cell cryopreservation after testing. To remove the precipitate, centrifuge the supernatant, 0.22 μ M pore size filter filtration and other methods. 3. please ensure that the cells are in good condition before freezing, and the proportion of viable cells should be greater than 90%. 4. this product contains DMSO, and some cells are sensitive to DMSO. It is recommended to conduct pre experiment. 5. for some precious cells, it is recommended to use the conventional freezing solution containing FBS for comparison test at the same time, and then conduct formal freezing after confirming the performance. 6. after subpackaging, the frozen cells should be moved into the -80 º C ultra-low temperature refrigerator as soon as possible to reduce the storage time outside. 7. for your safety and health, please wear experimental clothes and disposable gloves. Usage method: 1. Cell cryopreservation (1) Take out the cell cryopreservation solution, wait for complete melting and mixing, and store at 4 ℃ for later use. (2) Adhesive cells: Remove the cell culture medium, gently wash the cells once with sterile 1 × PBS to remove residual serum, and then add an appropriate amount of trypsin digestion solution to digest the cells. After digestion is complete, immediately add an appropriate amount of serum containing cell culture medium to terminate trypsin, then gently blow the cells and appropriately disperse and resuspend them. Note: Do not over digest cells, it is best to blow them down just enough. Overdigested cells are usually not suitable for cryopreservation due to poor growth conditions. The process of blowing and resuspending should also be gentle, otherwise it may affect the survival rate of cells during recovery. Suspension cells: Start from step (3). (3) Transfer the cell suspension to a suitable centrifuge tube. (4) Cell counting, calculating the total number of cells and the required amount of cell cryopreservation solution. The freezing density of cells is generally 1 × 106-1 × 107 cells/mL. (6.7 is a superscript) （5）100-200 xg， Centrifuge for 5-10 minutes and discard the supernatant. The centrifugation speed and time depend on the cell type. (6) Add the calculated volume of cell cryopreservation solution, gently blow with a pipette to resuspend the cells, and adjust the cell density according to the cell type (usually 1 × 106 cells/mL or higher). (6 is a superscript) (7) Packaging. Divide the above cell suspension into 1.5 mL or 2 mL cell cryovials and label them accordingly. Note: If stored in a liquid nitrogen tank later, a cell cryopreservation tube that can be used for liquid nitrogen cryopreservation must be used. (8) Place the cell cryopreservation tube in a -80 º C freezer. Note: If it is intended to be stored in liquid nitrogen for a long time, it can be stored in a -80 º C refrigerator for 24 hours and then transferred to a liquid nitrogen tank. 2. Cell revival (1) Remove the cryovial from a -80 º C refrigerator or liquid nitrogen and immediately place it in a 37 º C water bath. Gently shake for less than 1 minute until a small amount of ice remains. (2) Transfer the above cell suspension to a 15 mL sterile centrifuge tube, add 5-10 mL of preheated complete culture medium, and gently mix. Note: For some cells with high recovery efficiency, the cell suspension can also be directly transferred to a centrifuge tube for the next step. (3) 100-200 × g, centrifuge for 5-10 min, centrifugation speed and time depend on cell type. (4) Settle the cells and carefully remove the supernatant. (5) Add an appropriate amount of preheated complete culture medium, gently blow it evenly, transfer it to a culture vessel, and place it in a cell culture incubator for cultivation. Scope of application： Cell cryopreservation

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