Freezing protocol
冷冻步骤
1. Wash the cells with warm PBS solution, aspirate the solution ａnd cover the cells with a solution containing trypsin ａnd EDTA (a thin liquid film is enough; the concentration should be evaluated for each cell line).
用温的 PBS 溶液洗涤细胞，吸取溶液，含有胰蛋白酶和 EDTA 的溶液覆盖细胞（薄薄的液层足够了，胰蛋白酶和 EDTA 的浓度需要根据细胞系确定）。
2. Incubate the cells for max. 3 – 5 min at 37 °C.
37℃孵育细胞 3-5 分钟。
3. Once the cells detach from the bottom, stop incubation by adding cell culture medium supplemented with serum ａnd slightly suspend cells using a pipette.
细胞从底部脱离之后，终止孵育，加入含有血清的培养基，用移液器轻轻地悬浮细胞。
4. Spin down the suspension (500 x g, 5 min) ａnd resuspend the pellet with medium containing serum.
离心细胞悬液（500 x g, 5 分钟），用含有血清的培养基重新悬浮。
5. Determine the cell number (using a Neubauer chamber).
细胞计数。
6. Spin down the cells for 5 min at 500 x g ａnd discard the supernatant. Resuspend the pellet with an adequate volume of cell culture medium containing serum.
离心细胞悬液（500 x g, 5 分钟），去除上清液，用适量体积的含有血清的培养基重新悬浮细胞。
7. Mix the cell suspension 1:1 with freezing medium (60 % medium, 20 % FCS, 20 % DMSO) ａnd transfer it in Cryo.STM. For freezing in Cryo.STM the concentration of cells should be 1 – 5 x 106 cells / ml.
2/4 以 1:1 体积比混合细胞悬液和冻存液（60%培养基，20%胎牛血清，20% DMSO），然后转移到 Cryo.STM 冻存管中。冻存的细胞密度为 1-5×106 个/毫升。
8. Cryo.STM containing cells should be frozen at a cooling rate of -1 K / min. This can be achieved by placing them into an isopropanol-filled chamber at -70 °C. If other types of samples are contained, Cryo.s™ may be frozen directly at -20 °C, -70 °C or in the gas phase of liquid nitrogen. In order to assure even freezing of the sample, 4 ａnd 5 ml Cryo.s™ should be frozen at -20 °C overnight before transferring them to -70 °C or to the gas phase of liquid nitrogen.
含有细胞的 Cryo.STM 冻存管建议以-1 K / min 的速率降温，可以将冻存管置于-70℃含有异丙醇的容器中。如果Cryo.STM冻存管含有其他样品，可以直接放置在-20℃，-70℃ 或者液氮的气相。为了确保样品冷冻均匀，4 ml 和 5 ml 的 Cryo.STM冻存管需要先置于在-20℃冰箱过夜，然后再转移到-70℃或者液氮的气相。
9. Then transfer the Cryo.STM into the nitrogen tank. To avoid contamination (e. g. mycoplasma) ａnd due to safety precautions it is recommended to store the Cryo.STM in the gas phase above ａnd not in the liquid nitrogen.
然后转移 Cryo.STM 冻存管到液氮罐。为了避免污染（如支原体）和安全考虑，请将 Cryo.STM 冻存管置于液氮的气相，切勿置于液相。

Thawing protocol
解冻步骤
1. Immediately after removing them out of the nitrogen tank the frozen cells are thawed in about 1 – 2 min brandishing the Cryo.STM in a water bath at 37 °C. The thawing process should be performed as fast as possible.
从液氮罐取出冻存的细胞后立即置于 37℃水浴摇晃 Cryo.STM 冻存管 1～2 分钟。解冻过程需要越快越好。
2. Transfer the thawed cell suspension into a 15 ml tube ａnd mix it immediately with copious amounts of cell culture medium containing serum.
转移解冻的细胞悬液于 15 mL 离心管，立即用大量的含有血清的细胞培养基混匀。 3/4
3. After spinning down the cells (500 x g, 5 min) discard the supernatant ａnd resuspend the pellet in an appropriate cell culture medium supplemented with serum ａnd transfer it into one or more cell culture flasks.
500 x g离心细胞5 分钟，去除上清液，用适量的含有血清的细胞培养基重新悬浮细胞，然后转移到细胞培养瓶。
4. Follow the recommended cell concentration for seeding.
按照建议的细胞浓度接种。
5. During the next 12 hours cells should rest.
接下来的 12 小时细胞进入静默期。
6. A change of medium is recommended after 24 resp. 48 hours.
间隔 24 小时和 48 小时更换培养基

Safety advisory for working with Cryo.STM
Cryo.STM冻存管安全操作建议

Cryo.STM tubes are intended for sample storage exclusively in the gas phase over liquid nitrogen or in freezers! If Cryo.STM are stored in the liquid phase, nitrogen can seep into the tubes. Then upon thawing the vaporizing nitrogen can generate high pressure, ultimately resulting in an explosion, as well as the release of any infectious material.
Cryo.STM冻存管用来存储样品，只能置于液氮气相或冰箱。如果 Cryo.STM 冻存管浸没于液氮液相，液氮可能渗入冻存管。因此，解冻时，蒸发的液氮产生高压力，*终导致冻存管炸裂，并且还将导致感染物质释放。
Always take appropriate personal safety measures when working with Cryo.STM, including wearing safety clothing, using goggles ａnd working at a safety laboratory bench.
因此，操作 Cryo.STM 冻存管时需要佩戴合适的个人安全防护措施，如穿戴安全防护服、佩戴护目镜、在安全橱操作。
When undertaking cryogenic preservation, Cryo.STM must be evenly exposed to freezing temperatures. Uneven temperature exposures can cause formation of ice plugs (i. e. at tube 4/4 top) that inhibit the expansion of freezing liquid (i. e. at tube bottom), resulting in dangerous high pressure ａnd subsequent harm or damage of tubes.
进行冷冻保存时，Cryo.STM 冻存管需要缓慢地降温至冷冻温度。如果不是缓慢地降温到冷冻温度，将导致冰塞形成（如在冻存管顶部），冰塞将阻碍液体形成凝固（如在冻存管顶部），将导致高压力危险和后续的爆管风险。
Never exceed maximum working volumes as specified.
不要超过标识的工作体积

原创作者：北京鼎国昌盛

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