4 replies to this topic

[SAPkinase]

hai,

I want to work with intestinal epithelial cells. I tried isolating them using protocols in literature. Could get a good population of viable cells (98% by trypan blue staining). but after i seed them in poly lysine coated plates, the next day wat i could see is only floating cells with complete acidified medium. I use DMEM/F12+10% FCS. I use penicillin strep and gentamycin, so no contamination is seen. there are some complicated additives written in literature without stating the reason. Is there anyone who can help me with  a solution. I am stuck up....

Thanks in advance

[bob1]

It sounds like you have a lot of necrosis of the cells going on.  I suggest trying different coatings for your plates and making sure that you are not over-seeding (too high a density).  

As primary cells also like to have a whole heap of different growth factors around compared to immortalised cells, you should also try the additives that the papers suggest.  Another way to do this is to create a "feeder layer" or to "condition" your medium.  Check out R.I Freshney's "Culture of Animal Cells" for more information.

Incidentally:  I think you meant to say "I am stuck" rather than "I am stuck up", which means that you are arrogant, not unable to come up with a solution.

[genehunter]

I believe a line has been established by a  group in Canada.

[T2R]

Hi SAPkinase!I'm trying to work on intestinel epithelial cells too (IN MICE),but with really poor results...do you mind to send me the protocol you are using? tHANK YOU SOOOOOOOOOOOOO MUCH! :D

[rhombus]

SAPkinase, on Feb 14 2009, 01:12 PM, said:

hai,

I want to work with intestinal epithelial cells. I tried isolating them using protocols in literature. Could get a good population of viable cells (98% by trypan blue staining). but after i seed them in poly lysine coated plates, the next day wat i could see is only floating cells with complete acidified medium. I use DMEM/F12+10% FCS. I use penicillin strep and gentamycin, so no contamination is seen. there are some complicated additives written in literature without stating the reason. Is there anyone who can help me with  a solution. I am stuck up....

Thanks in advance

Dear SAPkinase,

"complete acidified media"....there are really only 4 causes this can be attributed to:-

a) Bacterial contamination....if the media is acidified and it is due to bacteria, then you should see them under phase contrast microscopy.
CO2 concentration in your incubator is massively too high....and incubator display may indicate 5%, but it may infact be 20%....but Fyrite it to make sure.
c) As bob1 quite rightly says.....seeding density may be too high.
d) I do not do intestinal primary culture....but one question would be "do these particular cells release more acid than other primary cells"?
If that was the case then the acidification of the media could be controlled by adding less cells AND more buffering capacity in your cell culture media. Under normal conditions as the adherent cells become more confluent and release acid, they detach from the surface if they are not refeed with media OR passaged.

Hope this helps

Kindest regards

Rhombus.