4 replies to this topic

[miraclestrain]

Hi All,

I borrowed MDCK cells from my prof, and I started culturing them from passage 25 to passage 35, I am using them for viral studies.

When i stain the cells i see vacuolation. This is what I do,

Grow them on slides for 24-32 hours, wash them with PBS pH 7.2
Then fix them using 80% acetone or 80% methanol,
Then stain them using hematoxylin then eosin with steps involving dehydration, I see vacuolation even before dehydration using ethanol.

THis means that there is problem in the cells can someone helo me out with this problem, what should I do with the vacuolation?

I use DMEM as growth medium and have dissolved ampicillin.

Please help

Thanks
MS

[bob1]

Do you have any pictures? Are you sure it is vacuolation? Are the cells senescent?

[miraclestrain]

yes i do have pics,

i will attach two of them, pls have a look. I will attach few more in sometime.

Please comment on them

MS

Attached File(s)

•  minisee0004.jpg (276.97K)
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•  minisee0006.jpg (346.81K)
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[bob1]

Your cultures are pretty confluent, it could be that the cells are senescent, though I have never worked with the line so I don't know if they are contact inhibited.

[LostintheLab]

They aren't contact inhibited, but yours seem too confluent. They can form domes and can start to become polarised epithelial at high cell densities (if I remember correctly)
http://www.atcc.org/ATCCAdvancedCatalogSea...logy#aProp90e6a

the link above is to the ATCC which has a picture and their recommendations for culture conditions.
Do you have any from an lower passage number ?- you have got a fairly high passage number there.maybe try a "younger" cell and see if you have the same problems.