I am using A549 cell line and i had made stocks in Nov 2017 and stored in -80. When I revived them back they are growing at a very slow pace. As doubling time in 22 hrs approx.But in this case I dont know why it is taking so long to revive. Earlier the cells use to grow very fast overnight.
What can be the probable reason?
Also I would like to know how much amount of complete medium should be added upon the cell thawed during revival? Is there any particular amount of media to be added? I have seen in one of the video that people have used 9 ml media with 1 ml cells +FBS+DMSO mixture upon revival.
In most of the video and articles speed of cell thaw should be fast and cell freezing should be slow. Although we don't have things like Mr.Frosty so we use to freeze the cells in -80.
Can anybody help me about the protocol to freeze cells in absence of Mr. Frosty. I think because of absence of mr. frosty or some loop hole during the freezing procedure is making the cells to grow so slow.
Hi Debaashish,
From what it sounds like, your cell viability isn't that high. I've had this problem with my 3T3-L1 cell lines and it's taken a while for them to grow. Typically after freeze thawing the cells are very sensitive and can die much more easily. I would try being as gentle as possible with the cells.
Normally I thaw the cells in a 37C water bath until half of the ice crystals are melted and then proceed to transfer the media into a T25 flask with pre-warmed (37C) full culture media and gently distribute the cells evenly. After about 4-24 hours your cells should start to adhere to the flask (if adhering cells) and then you can replace the media to remove the DMSO.
Not sure about freezing the cells without a Mr. Frosty, but I would try to invest in one. It does help a great deal and increases cell viability.
Good Luck
Dear Andrew
Please give detailed procedure of freezing the cells. Thawing procesure is clear.
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