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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(wen)的(de)(de) PBS 溶(rong)液(ye)洗滌(di)細胞，吸取溶(rong)液(ye)，含有胰蛋白酶和 EDTA 的(de)(de)溶(rong)液(ye)覆(fu)蓋細胞（薄(bo)薄(bo) 的(de)(de)液(ye)層足(zu)夠了(le)，胰蛋白酶和 EDTA 的(de)(de)濃度需要(yao)根據細胞系(xi)確(que)定）。
2. Incubate the cells for max. 3 – 5 min at 37 °C.
37℃孵育(yu)細胞(bao) 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(xi)胞從底部脫離之后，終止孵育，加入含(han)有(you)血清的培養基，用移液(ye)器(qi)輕(qing)輕(qing)地懸浮細(xi)胞。
4. Spin down the suspension (500 x g, 5 min) and resuspend the pellet with medium containing serum.
離心細(xi)胞懸(xuan)液（500 x g, 5 分鐘），用含有血清的培養基(ji)重新懸(xuan)浮。
5. Determine the cell number (using a Neubauer chamber).
細胞計數。
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(xi)胞懸液（500 x g, 5 分鐘），去除上清液，用適量體積的含(han)有血清的培養基(ji)重(zhong)新 懸浮(fu)細(xi)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(bi)混合細(xi)胞懸液和凍存液（60%培養(yang)基，20%胎牛(niu)血清，20% DMSO），然 后轉移到 Cryo.STM 凍存管中。凍存的細(xi)胞密度為 1-5×106 個/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(han)有(you)細胞的(de) Cryo.STM 凍存(cun)管(guan)建(jian)議以-1 K / min 的(de)速率降溫，可(ke)以將(jiang)凍存(cun)管(guan)置于-70℃含(han) 有(you)異(yi)丙醇的(de)容器中。如果(guo)Cryo.STM凍存(cun)管(guan)含(han)有(you)其(qi)他樣品，可(ke)以直接(jie)放置在-20℃，-70℃ 或者液(ye)氮的(de)氣(qi)相。為了(le)確(que)保(bao)樣品冷凍均勻，4 ml 和(he) 5 ml 的(de) Cryo.STM凍存(cun)管(guan)需要先置于 在-20℃冰箱過夜，然后(hou)再轉(zhuan)移到-70℃或者液(ye)氮的(de)氣(qi)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.
然后轉移 Cryo.STM 凍(dong)存管到液氮(dan)罐。為了避(bi)免(mian)污染（如支原(yuan)體）和安全考(kao)慮，請將 Cryo.STM 凍(dong)存管置(zhi)于液氮(dan)的(de)氣相，切勿置(zhi)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(cong)液氮罐取出凍(dong)(dong)存(cun)的細胞后(hou)立即置于 37℃水浴搖晃 Cryo.STM 凍(dong)(dong)存(cun)管 1～2 分鐘。解(jie)凍(dong)(dong) 過程需要(yao)越快(kuai)越好(hao)。
2. Transfer the thawed cell suspension into a 15 ml tube and mix it immediately with copious amounts of cell culture medium containing serum.
轉移解凍的細胞懸(xuan)液(ye)于 15 mL 離心(xin)管(guan)，立即用大量的含有(you)血清的細胞培養基混(hun)勻(yun)。 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離心細胞5 分鐘，去除上清液(ye)，用適(shi)量的含有血清的細胞培養(yang)基重新(xin)懸浮細胞， 然后轉移(yi)到細胞培養(yang)瓶(ping)。
4. Follow the recommended cell concentration for seeding.
按照(zhao)建議的細(xi)胞濃度接(jie)種。
5. During the next 12 hours cells should rest.
接下來的 12 小時細(xi)胞進入靜默期。
6. A change of medium is recommended after 24 resp. 48 hours.
間隔 24 小時和(he) 48 小時更換(huan)培養基
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(dong)(dong)存(cun)管(guan)用來存(cun)儲(chu)樣品，只能(neng)置(zhi)于液(ye)氮(dan)氣相(xiang)或冰箱。如果 Cryo.STM 凍(dong)(dong)存(cun)管(guan)浸沒于液(ye) 氮(dan)液(ye)相(xiang)，液(ye)氮(dan)可能(neng)滲入凍(dong)(dong)存(cun)管(guan)。因此(ci)，解凍(dong)(dong)時，蒸(zheng)發的液(ye)氮(dan)產生高壓(ya)力，最終導(dao)致凍(dong)(dong)存(cun)管(guan)炸 裂，并且還將導(dao)致感染物質釋放。
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(ci)，操作(zuo) Cryo.STM 凍存管時需要佩(pei)戴(dai)合適(shi)的個人安(an)(an)全防護(hu)(hu)措(cuo)施，如穿戴(dai)安(an)(an)全防護(hu)(hu)服、佩(pei) 戴(dai)護(hu)(hu)目鏡、在安(an)(an)全櫥操作(zuo)。
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.
進行冷(leng)(leng)凍(dong)(dong)(dong)(dong)(dong)保存時，Cryo.STM 凍(dong)(dong)(dong)(dong)(dong)存管(guan)(guan)需要緩慢(man)地降溫(wen)至冷(leng)(leng)凍(dong)(dong)(dong)(dong)(dong)溫(wen)度(du)(du)。如果不是(shi)緩慢(man)地降溫(wen)到冷(leng)(leng) 凍(dong)(dong)(dong)(dong)(dong)溫(wen)度(du)(du)，將導(dao)致冰塞(sai)形成（如在凍(dong)(dong)(dong)(dong)(dong)存管(guan)(guan)頂(ding)部），冰塞(sai)將阻(zu)礙液體形成凝(ning)固（如在凍(dong)(dong)(dong)(dong)(dong)存管(guan)(guan)頂(ding) 部），將導(dao)致高壓力危險和后續的爆管(guan)(guan)風險。
Never exceed maximum working volumes as specified.
不要超過(guo)標識的(de)工作體積
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