Caesar

介绍 Introduction

The thawing procedure is stressful to frozen cells, and using good technique and working quickly ensures that a high proportion of the cells survive the procedure. As with other cell culture procedures, we recommend that you closely follow the instructions provided with your cells and other reagents for best results. 解冻过程对冷冻细胞来说是紧张的，使用好的技术和快速的工作可以确保很大比例的细胞存活下来。与其他细胞培养程序一样，我们建议您严格按照细胞和其他试剂提供的说明进行操作，以获得最佳效果

• Thaw frozen cells rapidly (< 1 minute) in a 37°C water bath. 在摄氏37度的水浴中快速解冻冻住的细胞(< 1分钟)

• Dilute the thawed cells slowly, using pre-warmed growth medium. 用预先加热的培养基缓慢稀释解冻的细胞

• Plate thawed cells at high density to optimize recovery. 平板高密度解冻细胞以优化回收率

• Always use proper aseptic technique and work in a laminar flow hood. 始终使用适当的无菌操作，并在层流罩内工作

• Always wear personal protective equipment, including a face mask or goggles. Cryovials stored in liquid-phase present a risk of explosion when thawed. 始终佩戴个人防护装备，包括面罩或护目镜。液相储存的冰冻卵在解冻时有爆炸的危险

• Some freezing media contain DMSO, which is known to facilitate the entry of organic molecules into tissues. Handle reagents containing DMSO using equipment 一些冷冻介质含有 DMSO，这是众所周知的促进有机分子进入组织。使用设备处理含有 DMSO 的试剂

实验材料 Experimental material

• Cryovial containing frozen cells 含有冷冻细胞的冷冻瓶

• Complete growth medium, pre-warmed to 37°C 全生长培养基，预热至摄氏37度

• Disposable, sterile centrifuge tubes 一次性无菌离心管

• Water bath at 37°C 摄氏37度水浴

• 70% ethanol 70% 乙醇

• Tissue-culture treated flasks, plates, or dishes 经组织培养处理的烧瓶、盘子或培养皿

Protocol for Thawing Frozen Cells 冷冻细胞解冻方案

视频 5: 复苏细胞 Video 5: resuscitating cells

视频5介绍了细胞解冻的最佳方法，且在此过程中不会对细胞造成不利影响。 Our scientists demonstrate how to carefully transfer cells from storage in liquid nitrogen to the incubator. Video 5 shows the best way to thaw cells without adversely affecting them in the process. Our scientists demonstrate how to carefully transfer cells from storage in liquid nitrogen to the incubator

The following protocol describes a 下面的协议描述了一个general procedure 一般程序 for thawing cryopreserved cells. 用来解冻冻存的细胞For detailed protocols, always refer to the cell-specific product insert. 对于详细的协议，始终参考特定于单元格的产品插入

1. Remove the cryovial containing the frozen cells from liquid nitrogen storage and 将装有冷冻细胞的冷冻小瓶从液氮贮存器中取出immediately 立即 place it into a 37°C water bath. 把它放入37 ° c 的水浴中

2. Quickly thaw the cells (< 1 minute) by gently swirling the vial in the 37°C water bath until there is just a small bit of ice left in the vial. 快速解冻细胞(小于1分钟) ，轻轻旋转小瓶在37 ° c 的水浴，直到只有一点点冰留在小瓶

3. Transfer the vial it into a laminar flow hood. Before opening, wipe the outside of the vial with 70% ethanol. 将小瓶放入层流罩中，开放前用70% 乙醇擦拭小瓶外部

4. Transfer the desired amount of pre-warmed complete growth medium appropriate for your cell line 转移适合细胞系的预热完全生长培养基的所需量dropwise 逐层的 into the centrifuge tube containing the thawed cells. 进入含有解冻细胞的离心管

5. Centrifuge the cell suspension at approximately 200 × g for 5–10 minutes. The actual centrifugation speed and duration varies depending on the cell type. 将细胞悬浮液以大约200 × g 离心5-10分钟。实际离心速度和持续时间取决于电池类型

6. After the centrifugation, check the clarity of supernatant and visibility of a complete pellet. Aseptically decant the supernatant without disturbing the cell pellet. 离心后，检查上清液的透明度和完整颗粒的可见度。无菌倾倒上清液，不干扰细胞颗粒

7. Gently resuspend the cells in complete growth medium, and transfer them into the appropriate culture vessel and into the recommended culture environment. 将细胞在完全培养基中轻轻复苏，并将其转移到适当的培养容器和推荐的培养环境中

注： Note:The appropriate flask size depends on the number of cells frozen in the cryovial, and the culture environment varies based on the cell and media type. 合适的烧瓶大小取决于冰冻细胞的数量，培养环境因细胞和培养基类型的不同而不同