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problem to stain just one cell line in coculture


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#1 tamarov2

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Posted 23 February 2009 - 04:31 AM

Hello.
I need to stain with an antibody one cell line and than coculture it with a 2nd cell line for 24hr. So after the staining of the 1st cell line (in PBS) I wash all the excess of the antibody, transfer it to a complete medium with 10% serum and than mix it with the 2nd cell line.
The problem is that the 2nd cell line also expresses the same target protein. So after the 24hr coculture, I can see ( in FACS, using appropiate gating) that the antibody stains also the 2nd cell line (which is not good for me).
Do you have any ideas how to avoid this? Meaning, are there conditions that I can use during the coculture that will minimze the antibody transfer from the 1st cell line to the second?
Thanks!

#2 genehunter

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Posted 23 February 2009 - 08:19 AM

Instead of using antibody, you may be able to label one cell line with some dye or Q-dot. Talk to tech support of Invitrogen.

#3 tamarov2

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Posted 23 February 2009 - 11:34 PM

Instead of using antibody, you may be able to label one cell line with some dye or Q-dot. Talk to tech support of Invitrogen.

Thanks for the quick reply. But I do need to use the antibody since it is functional (I use it as a blocking antibody). Thanks again, Tamar

#4 genehunter

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Posted 24 February 2009 - 07:22 AM

I thought you meant to "stain", but you are trying to block some surface proteins with this antibody.

Well, most interaction is reversible and antibody-antigen is among the list. Therefore redistribution is expected over a long time. On top of that, antibody/antigen complex can get endocytosed or protease degraded, new antigen may re-emerge over the same period. So I can see you will have to face multitude of problmes.

Solutions? there is no magic way. Chosing an antibody with higher affinity than the one you are using may help.

If you dont have any other choice, either you have to chose a different cell pair to work with, or reduce the time.

Affinity cross-linking/labeling in principle can help stablize the complex, but its not easy to do in reality.

#5 tamarov2

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Posted 25 February 2009 - 01:22 AM

Thanks a lot. you have been very helpful. I thought there is some easy way to overcome my problem but I understand there is not..
Thanks again, Tamar


I thought you meant to "stain", but you are trying to block some surface proteins with this antibody.

Well, most interaction is reversible and antibody-antigen is among the list. Therefore redistribution is expected over a long time. On top of that, antibody/antigen complex can get endocytosed or protease degraded, new antigen may re-emerge over the same period. So I can see you will have to face multitude of problmes.

Solutions? there is no magic way. Chosing an antibody with higher affinity than the one you are using may help.

If you dont have any other choice, either you have to chose a different cell pair to work with, or reduce the time.

Affinity cross-linking/labeling in principle can help stablize the complex, but its not easy to do in reality.






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