0.25%胰蛋白酶溶液（含EDTA，溶于PBS）

0.25%胰蛋白酶溶液（含EDTA，溶于PBS）现货供应，

细胞在培养瓶中培养至良好状态后灌满完全培养液并封好瓶口是细胞运输的最好办法。收到细胞回到自己的实验室后，先打开外包装，用75%酒精喷洒整个瓶消毒后放到超净台内，严格无菌操作，培养箱静置2-4小时。镜下观察：未超过80%汇合度时，可将瓶装的完全培养液移入废液缸中，加入6ml新鲜完全培养基，放入37℃、5%CO2孵箱培养；超过80%汇合度时，根据情况传代或者冻存，具体操作见细胞培养步骤。（注意发货的是密封培养瓶的话，放入培养箱培养记得培养瓶盖子拧松）

After the cells have been cultured in a flask to a good state, filling the entire culture medium and sealing the bottle mouth is the best way to transport the cells. After receiving the cells and returning to their own laboratory, the outer packaging was opened, the whole bottle was sprayed with 75% alcohol for sterilization, and then placed in the clean table, strictly aseptic operation, and the incubator stood for 2-4 hours. Microscopic observation: when the confluence does not exceed 80%, the bottled complete culture can be transferred into the waste tank, 6ml of fresh complete medium can be added, and placed in a 37°C, 5% CO2 incubator for culture; When the confluency exceeds 80%, passaging or cryopreservation, according to the situation, see the cell culture steps for specific operations. (Note that if the flask is sealed, remember to loosen the lid of the flask when you put it in the incubator)

0.25%胰蛋白酶溶液（含EDTA，溶于PBS）培养步骤
1）复苏细胞：将含有1mL细胞悬液的冻存管在37℃水浴中迅速摇晃解冻，加入5mL培养基混合均匀。在1000RPM条件下离心5分钟，弃去上清液，补加4-6mL完全培养基后吹匀。然后将所有细胞悬液加入培养瓶中培养过夜（或将细胞悬液加入6cm皿中），培养过夜。第二天换液并检查细胞密度；
2）细胞传代：如果细胞密度达80%-90%，即可进行传代培养
1、对于贴壁细胞，传代可参考以下方法：
1. 弃去培养上清，用不含钙、镁离子的PBS润洗细胞1-2次。
2. 加1-2ml消化液（0.25%Trypsin-0.53mM EDTA）于培养瓶中，置于37℃培养箱中消化1-2min，然后在显微镜下观察细胞消化情况，若细胞大部分变圆并脱落，迅速拿回操作台，轻敲几下培养瓶后加5ml以上含10%血清的完全培养基终止消化。
3.轻轻吹打细胞，完全脱落后吸出，在1000RPM条件下离心8-10分钟，弃去上清液，补加1-2mL培养液后吹匀。
4. 按5-6ml/瓶补加培养液，将细胞悬液按1：2到1：5的比例分到新的含5-6 ml培养液的新皿中或者瓶中。
PS：若客户收到2ml小管细胞，收到细胞后，用75%酒精喷洒整个管子消毒后放到超净台或安全柜内，严格无菌操作；将小管细胞转移至T25培养瓶或6cm培养皿，加入5ml左右完全培养基混匀，放入培养箱过夜培养后查看细胞密度：若密度未超过80%，换液继续培养，视情况传代或者冻存。若密度超过80%，可直接进行传代（方法同上）。

1) Thaw cells: Shake the cryovial containing 1 mL of cell suspension in a 37 °C water bath and add 5 mL of medium to mix well. Centrifuge at 1000 RPM for 5 min, discard the supernatant, add 4-6 mL of complete medium and blow well. Then add all cell suspensions to the flask and incubate overnight (or add the cell suspension to a 6 cm dish) and incubate overnight. The next day, feed and check cell density;
2) Cell passaging: If the cell density reaches 80%-90%, subculture can be performed
1. For adherent cells, the following methods can be referred to for passaging:
Discard the culture supernatant and rinse the cells 1-2 times with PBS without calcium and magnesium ions.
2. Add 1-2ml of digestion solution (0.25% Trypsin-0.53mM EDTA) to the culture flask, put it in a 37°C incubator for digestion for 1-2min, and then observe the cell digestion under the microscope, if most of the cells become round and fall off, quickly take it back to the operating table, and add more than 5ml of complete medium containing 10% serum to terminate the digestion after a few taps of the culture flask.
3. Gently pipette the cells, aspirate after they are completely detached, centrifuge at 1000RPM for 8-10 minutes, discard the supernatant, add 1-2mL of culture medium and blow well.
4. Add the culture medium at a rate of 5-6 ml/bottle, and divide the cell suspension into a new dish or bottle containing 5-6 ml of culture medium in a ratio of 1:2 to 1:5.
PS: If the customer receives 2ml of tubular cells, after receiving the cells, spray the entire tube with 75% alcohol to disinfect and put it in the ultra-clean table or safety cabinet, and strictly aseptic operation; Transfer the tubular cells to a T25 flask or 6cm dish, add about 5ml of complete medium to mix well, put it in the incubator overnight and check the cell density: if the density does not exceed 80%, change the liquid and continue to culture, passage or cryopreservation as appropriate. If the density exceeds 80%, it can be passaged directly (method as above).