为何即使在胶原酶IV的分离时间（37°C条件下30-60分钟，实际效果视特定批次而定）比分散酶更长的情况下，也最好使用IV型胶原酶而不是分散酶？
实际上，在基于饲养层的培养体系中，分散酶（2 mg/mL）需要37°C条件下进行15-25分钟的孵育。无饲养层的培养体系需2-3分钟的解离时间。分散酶是一种更强烈的酶，所以起效更快，但也意味着当收获PSC细胞团时，它们对后续的吹打更为敏感。在收获细胞团时，它们会被上下吸取数次，从而形成适当大小的团块。如果应用IV型胶原酶收获细胞，则由于细胞团更难分解，可施加更多次数的吹打，不过这也意味着细胞团不至于被解离成非常小的团块。如果采用分散酶收获细胞，它们需吹打的次数更少，用户也需要多加小心，不要将细胞团分散过度。两种酶的使用效果均不错，如果您拥有足够的经验，您可能更喜欢使用分散酶节省操作时间。但对于经验不足的用户，我们推荐您使用IV型胶原酶，因为它更为安全，过度吹打造成培养物损伤的可能性也更小。
你们提供哪些用于解离细胞的试剂？这些产品之间的主要区别是什么？
请通过此选择列表(https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/trypsin.html)来比较我们的细胞解离试剂。
How do I make the 1000X stock (100 U/µL) solution from Collagenase powder?
1. Add 1 mL Hanks' Balanced Salt Solution (HBSS) with calcium and magnesium directly to 1 g vial of Collagenase. Vortex gently to ensure complete dissolution. Transfer to a clean tube.
2. Determine volume of HBSS (with calcium and magnesium) required to bring collagenase solution to 100 U/µL (1000X stock solution). The activity is lot- specific. Rinse vial with this volume of HBSS (with calcium and magnesium), and combine. Filter sterilize 1000X stock solution with a low protein binding filtration unit.
Example: Assuming the lot you have purchased has an activity of 265 U/mg, this lot will have 265000 Units per mL when you reconstitute collagenase into HBSS (with calcium and magnesium) at 1 g/mL. In order to dilute 265000 U/L to 100000 U/mL (= 100 U/µL), you need to dilute the 1 g/mL enzyme solution 2.65 fold.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Why is collagenase type IV favored over dispase even though the dissociation with collagenase IV seems to take longer (between 30 and 60 min, depending from the lot, at 37 degrees C) compared to dispase?
Actually, in a feeder-based culture, dispase (2 mg/mL) should take about 15-25 min to work at 37 degrees C. Two to three minutes' dissociation time would apply to feeder-free cultures. Dispase is a more aggressive enzyme, so it works faster, but that also means that when the PSC clumps are harvested, they are more sensitive to being broken apart by trituration. Once the clumps are harvested, they should be pipetted up and down a few times to break up the clumps to the appropriate size. If the cells are harvested with collagenase type IV, they have to be pipetted more times because the clumps are harder to break up, but this means that there is less likelihood to break up the clumps into pieces that are too small. If the cells are harvested with dispase, they have to be pipetted fewer times, and care has to be taken to ensure that the clumps are not broken too much. Either enzyme is fine to use, and if you have enough experience, you may prefer to use dispase to save time. But for a less experienced user, we recommend using collagenase type IV as it is safer and you are less likely to ruin your culture by over-triturating.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
What reagents do you offer for cell dissociation, and what are the main differences between them?
Please use this selection chart that compares our cell dissociation reagents (https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/trypsin.html).
Find additional tips, troubleshooting help, and resources within ourMammalian Cell Culture Basics Support Center.
