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Freezing protocol
冷凍步驟
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).
用溫的 PBS 溶液洗滌細(xì)胞，吸取溶液，含有胰蛋白酶和 EDTA 的溶液覆蓋細(xì)胞（薄薄 的液層足夠了，胰蛋白酶和 EDTA 的濃度需要根據(jù)細(xì)胞系確定）。
2. Incubate the cells for max. 3 – 5 min at 37 °C.
37℃孵育細(xì)胞 3-5 分鐘。
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.
細(xì)胞從底部脫離之后，終止孵育，加入含有血清的培養(yǎng)基，用移液器輕輕地懸浮細(xì)胞。
4. Spin down the suspension (500 x g, 5 min) and resuspend the pellet with medium containing serum.
離心細(xì)胞懸液（500 x g, 5 分鐘），用含有血清的培養(yǎng)基重新懸浮。
5. Determine the cell number (using a Neubauer chamber).
細(xì)胞計(jì)數(shù)。
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.
離心細(xì)胞懸液（500 x g, 5 分鐘），去除上清液，用適量體積的含有血清的培養(yǎng)基重新 懸浮細(xì)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 體積比混合細(xì)胞懸液和凍存液（60%培養(yǎng)基，20%胎牛血清，20% DMSO），然 后轉(zhuǎn)移到 Cryo.STM 凍存管中。凍存的細(xì)胞密度為 1-5×106 個(gè)/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.
含有細(xì)胞的 Cryo.STM 凍存管建議以-1 K / min 的速率降溫，可以將凍存管置于-70℃含 有異丙醇的容器中。如果Cryo.STM凍存管含有其他樣品，可以直接放置在-20℃，-70℃ 或者液氮的氣相。為了確保樣品冷凍均勻，4 ml 和 5 ml 的 Cryo.STM凍存管需要先置于 在-20℃冰箱過(guò)夜，然后再轉(zhuǎn)移到-70℃或者液氮的氣相。
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.
然后轉(zhuǎn)移 Cryo.STM 凍存管到液氮罐。為了避免污染（如支原體）和安全考慮，請(qǐng)將 Cryo.STM 凍存管置于液氮的氣相，切勿置于液相。
Thawing protocol
解凍步驟
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.
從液氮罐取出凍存的細(xì)胞后立即置于 37℃水浴搖晃 Cryo.STM 凍存管 1～2 分鐘。解凍 過(guò)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.
轉(zhuǎn)移解凍的細(xì)胞懸液于 15 mL 離心管，立即用大量的含有血清的細(xì)胞培養(yǎng)基混勻。 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離心細(xì)胞5 分鐘，去除上清液，用適量的含有血清的細(xì)胞培養(yǎng)基重新懸浮細(xì)胞， 然后轉(zhuǎn)移到細(xì)胞培養(yǎng)瓶。
4. Follow the recommended cell concentration for seeding.
按照建議的細(xì)胞濃度接種。
5. During the next 12 hours cells should rest.
接下來(lái)的 12 小時(shí)細(xì)胞進(jìn)入靜默期。
6. A change of medium is recommended after 24 resp. 48 hours.
間隔 24 小時(shí)和 48 小時(shí)更換培養(yǎng)基
Safety advisory for working with Cryo.STM
Cryo.STM凍存管安全操作建議
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凍存管用來(lái)存儲(chǔ)樣品，只能置于液氮?dú)庀嗷虮?。如?Cryo.STM 凍存管浸沒于液 氮液相，液氮可能滲入凍存管。因此，解凍時(shí)，蒸發(fā)的液氮產(chǎn)生高壓力，最終導(dǎo)致凍存管炸 裂，并且還將導(dǎo)致感染物質(zhì)釋放。
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 凍存管時(shí)需要佩戴合適的個(gè)人安全防護(hù)措施，如穿戴安全防護(hù)服、佩 戴護(hù)目鏡、在安全櫥操作。
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.
進(jìn)行冷凍保存時(shí)，Cryo.STM 凍存管需要緩慢地降溫至冷凍溫度。如果不是緩慢地降溫到冷 凍溫度，將導(dǎo)致冰塞形成（如在凍存管頂部），冰塞將阻礙液體形成凝固（如在凍存管頂 部），將導(dǎo)致高壓力危險(xiǎn)和后續(xù)的爆管風(fēng)險(xiǎn)。
Never exceed maximum working volumes as specified.
不要超過(guò)標(biāo)識(shí)的工作體積
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