I had prepared mouse adult fibroblasts from Wild type and Knock-out line. They were proliferating very well. I immortilzed them with SV40 T large antigen vector at passage 4 and split them till 10 passages and again they were ok. Then I freezed them with by using 10% DMSO, 40% DMEM and 50% FBS. I took one of the vial and put them in culture after some time but now cells are not behaving good. there are fibroblasts cells adherent but there are another type of cells are also there and they are rounded. In Wt they are lot of approx. 80% and in KO only 2-5% . I am bit surprized because I am working on two KO lines together and other line is working very well, same procedure,same protocols, same handling time. Can any one please help me that what could be the possible reason for that?
I checked from two vials but I still have more so may be I can try again. And now I have 2ud preparation also and this time I will more keep in mind for this freezing and thawing cycle.
Thanks for your help :)
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